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Illumina Inc preparation kit
Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 288 article reviews
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Centrifugation:

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: After centrifugation, the supernatant was extracted twice with phenol-chloroform. .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina).

Construct:

Article Title: Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium
Article Snippet: We obtained 259,087 fragment reads and 287,202, 308,517, and 318,239 paired-end reads with insert sizes of 8 kbp, 20 kbp, and 30 kbp, respectively. .. For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively. .. We obtained 2,201,763 paired-end reads, with an insert size of 800 bp.

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Gene Set Enrichment Analysis (GSEA) of the 177 differentially expressed targets included testing against GO BP, Biocarata, Kegg, and Reactome gene sets. .. Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Poly-A containing mRNA from the total RNA is isolated using poly-T oligo-attached magnetic bead selection.

Real-time Polymerase Chain Reaction:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina). .. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina).

Cell Culture:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: N. cycadae strain WK-1 keeps the potential of cell differentiation and can be cultured at a high density on an agar plate or in liquid medium using a modified Detmer’s medium ( ) or BG-11 medium ( ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Expressing:

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome
Article Snippet: In the type II CCA, the CcaS-CcaR system directly regulates the expression of the rod linker gene of phycoerythrin and, in several species, the hydrophobic rod-core linker of phycocyanin ( ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Modification:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: N. cycadae strain WK-1 keeps the potential of cell differentiation and can be cultured at a high density on an agar plate or in liquid medium using a modified Detmer’s medium ( ) or BG-11 medium ( ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Derivative Assay:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively. .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Flow Cytometry:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Finally, the products are purified and PCR-enriched to create the double-stranded cDNA library.

Inverse PCR:

Article Title: Complete Genome Sequence of the Bacteriochlorophyll b-Producing Photosynthetic Bacterium Blastochloris viridis
Article Snippet: In 2004, we sequenced the photosynthetic gene cluster of B. viridis using an inverse PCR and Sanger method, and some genes in the cluster have already been deposited at GenBank (accession no. AB738834). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Ligation:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Poly-A containing mRNA from the total RNA is isolated using poly-T oligo-attached magnetic bead selection.

Infection:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: The hormogonia exhibit temporally the chemotactic and/or phototactic gliding motility involved in the infection processes toward the host plant tissue ( ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: As a first step to investigate the genetic base of fungal virulence factors and understand the mechanism of infection, we have assembled the draft genome sequence of P. longicolla type strain TWH P74 (ATCC 60325), which was originally isolated by Hobbs et al. from soybean seed in Ohio in 1983 ( ). .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols.

Cell Differentiation:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: N. cycadae strain WK-1 keeps the potential of cell differentiation and can be cultured at a high density on an agar plate or in liquid medium using a modified Detmer’s medium ( ) or BG-11 medium ( ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Generated:

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: Genomic DNA of type strain TWH P74 was extracted from a 4-day-old culture and used to generate mate-pair and paired-end libraries with insert sizes of approximately 4 kb and 500 bp, respectively. .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols. .. Libraries were sequenced in separate lanes on an Illumina HiSeq 2500 sequencer using a TruSeq SBS sequencing kit (version 3, Illumina) at the Genomics Core Facility, Purdue University.

Isolation:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: The isolation of cyanobacterial genomic DNA (gDNA) and whole-genome sequencing were performed using a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq reagent kit version 3 (600 cycles; Illumina), as shown in previous reports ( , ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: As a first step to investigate the genetic base of fungal virulence factors and understand the mechanism of infection, we have assembled the draft genome sequence of P. longicolla type strain TWH P74 (ATCC 60325), which was originally isolated by Hobbs et al. from soybean seed in Ohio in 1983 ( ). .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols.

Article Title: Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755
Article Snippet: To understand the molecular diversity of the genus Leptolyngbya , we isolated Leptolyngbya sp. strain NIES-3755 from soil at the Toyohashi University of Technology, Toyohashi, Aichi, Japan, and determined the complete genome sequence. .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome
Article Snippet: The cyanobacterium Geminocystis sp. strain NIES-3709 accumulates a larger amount of phycoerythrin than the related NIES-3708 strain does, although the two strains are isolated from the same freshwater stream. .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of the Bacteriochlorophyll b-Producing Photosynthetic Bacterium Blastochloris viridis
Article Snippet: Blastochloris viridis is a member of anoxygenic phototrophic bacteria in the phylum Proteobacteria (α-2 subclass) ( , ), which are often called “purple bacteria.” This bacterium is unique because it produces bacteriochlorophyll (BChl) b which has an absorption maximum in the near-infrared light region ( , ), whereas most isolated purple bacteria produce BChl a ( , ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Gene Set Enrichment Analysis (GSEA) of the 177 differentially expressed targets included testing against GO BP, Biocarata, Kegg, and Reactome gene sets. .. Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Poly-A containing mRNA from the total RNA is isolated using poly-T oligo-attached magnetic bead selection.

Sequencing:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: The isolation of cyanobacterial genomic DNA (gDNA) and whole-genome sequencing were performed using a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq reagent kit version 3 (600 cycles; Illumina), as shown in previous reports ( , ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively.

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: As a first step to investigate the genetic base of fungal virulence factors and understand the mechanism of infection, we have assembled the draft genome sequence of P. longicolla type strain TWH P74 (ATCC 60325), which was originally isolated by Hobbs et al. from soybean seed in Ohio in 1983 ( ). .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols.

Article Title: Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium
Article Snippet: We obtained 259,087 fragment reads and 287,202, 308,517, and 318,239 paired-end reads with insert sizes of 8 kbp, 20 kbp, and 30 kbp, respectively. .. For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively. .. We obtained 2,201,763 paired-end reads, with an insert size of 800 bp.

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome
Article Snippet: To explore the molecular basis of the different cellular phycoerythrin contents in the two strains, we performed whole-genome sequencing of the NIES-3709 strain using the MiSeq (Illumina) system. .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of the Bacteriochlorophyll b-Producing Photosynthetic Bacterium Blastochloris viridis
Article Snippet: Genome sequencing was performed using the MiSeq system (Illumina). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina). .. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina).

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Prior to sequencing, FAIRE DNA was analysed and quantified by running 1/25 of the FAIRE material on a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina).

Sonication:

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Samples were then sonicated for 16 sessions of 1 min (30 sec on/30 sec off) using a bioruptor (Diagenode) at max intensity, at 4°C. .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina).

ChIP-sequencing:

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Prior to sequencing, FAIRE DNA was analysed and quantified by running 1/25 of the FAIRE material on a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina). .. Single-read sequencing (36bp) was performed on a Genome Analyzer II (Illumina).

RNA Sequencing Assay:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Paragraph title: RNA Sequencing (bone marrow) ... Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA).

Article Title: m6A facilitates hippocampus-dependent learning and memory through Ythdf1
Article Snippet: Paragraph title: RNA-seq. ... The RNA libraries were prepared using Truseq stranded mRNA sample preparation kit (Illumina) according to manufacturer’s protocol.

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Paragraph title: RNA sequencing (RNA-seq) ... Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina).

Random Primed:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Poly-A containing mRNA from the total RNA is isolated using poly-T oligo-attached magnetic bead selection.

Size-exclusion Chromatography:

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Samples were then sonicated for 16 sessions of 1 min (30 sec on/30 sec off) using a bioruptor (Diagenode) at max intensity, at 4°C. .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina).

Purification:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. RNA was chemically fragmented and random primed prior to reverse transcription and cDNA generation. cDNA fragments then undergo end repair, the addition of a single ‘A’ base to the 3' end, and ligation of adapters.

Article Title: m6A facilitates hippocampus-dependent learning and memory through Ythdf1
Article Snippet: Total RNA from WT littermate control and Ythdf1 -KO mouse (8–16 weeks, male) hippocampus was extracted using Trizol (Invitrogen) and isopropanol precipitation. mRNA extraction was performed by poly(A)+ RNA selection once using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen). .. The RNA libraries were prepared using Truseq stranded mRNA sample preparation kit (Illumina) according to manufacturer’s protocol.

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: The resulting correlation was significant (p < 2E-16; adjusted R2 =0.2221) .. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina). .. Samples were individually indexed for pooling using a dual-index strategy.

Polymerase Chain Reaction:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: The isolation of cyanobacterial genomic DNA (gDNA) and whole-genome sequencing were performed using a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq reagent kit version 3 (600 cycles; Illumina), as shown in previous reports ( , ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively. .. The obtained reads were cleaned by removing low-quality ( < 25 Phred score) and short ( < 100-bp) reads.

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: Genomic DNA of type strain TWH P74 was extracted from a 4-day-old culture and used to generate mate-pair and paired-end libraries with insert sizes of approximately 4 kb and 500 bp, respectively. .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols. .. Libraries were sequenced in separate lanes on an Illumina HiSeq 2500 sequencer using a TruSeq SBS sequencing kit (version 3, Illumina) at the Genomics Core Facility, Purdue University.

Article Title: Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755
Article Snippet: Whole-genome sequencing of the NIES-3755 strain was performed using MiSeq sequencer (Illumina, San Diego, CA) and in silico finishing software as reported previously ( ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. 300 bp of each end of the libraries were sequenced on the MiSeq instrument with the MiSeq reagent kit v3 (600-cycles; Illumina).

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation
Article Snippet: The libraries were sequenced on the GS FLX+ instrument, yielding 309,976 shotgun reads and 222,244 paired-end reads. .. For MiSeq, an 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. The libraries were sequenced on the MiSeq instrument with the MiSeq reagent kit version 2 (500 cycles; Illumina), which yielded 668,946 paired-end reads and 539,927 mate-pair reads.

Article Title: Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium
Article Snippet: We obtained 259,087 fragment reads and 287,202, 308,517, and 318,239 paired-end reads with insert sizes of 8 kbp, 20 kbp, and 30 kbp, respectively. .. For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively. .. We obtained 2,201,763 paired-end reads, with an insert size of 800 bp.

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. RNA was chemically fragmented and random primed prior to reverse transcription and cDNA generation. cDNA fragments then undergo end repair, the addition of a single ‘A’ base to the 3' end, and ligation of adapters.

Periodic Counter-current Chromatography:

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation
Article Snippet: Interestingly, the antenna gene sets that are regulated by CcaS or RcaE are different among the analyzed cyanobacterial species Synechocystis sp. PCC 6803, Nostoc punctiforme ATCC 29133, and Fremyella diplosiphon . .. For MiSeq, an 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

cDNA Library Assay:

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. RNA was chemically fragmented and random primed prior to reverse transcription and cDNA generation. cDNA fragments then undergo end repair, the addition of a single ‘A’ base to the 3' end, and ligation of adapters.

Chromatin Immunoprecipitation:

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Prior to sequencing, FAIRE DNA was analysed and quantified by running 1/25 of the FAIRE material on a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina).

Software:

Article Title: Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755
Article Snippet: Whole-genome sequencing of the NIES-3755 strain was performed using MiSeq sequencer (Illumina, San Diego, CA) and in silico finishing software as reported previously ( ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Selection:

Article Title: m6A facilitates hippocampus-dependent learning and memory through Ythdf1
Article Snippet: Total RNA from WT littermate control and Ythdf1 -KO mouse (8–16 weeks, male) hippocampus was extracted using Trizol (Invitrogen) and isopropanol precipitation. mRNA extraction was performed by poly(A)+ RNA selection once using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen). .. The RNA libraries were prepared using Truseq stranded mRNA sample preparation kit (Illumina) according to manufacturer’s protocol.

Sample Prep:

Article Title: Draft Genome Sequence of the Nitrogen-Fixing and Hormogonia-Inducing Cyanobacterium Nostoc cycadae Strain WK-1, Isolated from the Coralloid Roots of Cycas revoluta
Article Snippet: The isolation of cyanobacterial genomic DNA (gDNA) and whole-genome sequencing were performed using a MiSeq sequencer (Illumina, San Diego, CA) with the MiSeq reagent kit version 3 (600 cycles; Illumina), as shown in previous reports ( , ). .. An 800-bp paired-end library and an 8-kbp mate pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate pair sample preparation kit (Illumina), respectively. .. The obtained reads were cleaned by removing low-quality ( < 25 Phred score) and short ( < 100-bp) reads.

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: Genomic DNA of type strain TWH P74 was extracted from a 4-day-old culture and used to generate mate-pair and paired-end libraries with insert sizes of approximately 4 kb and 500 bp, respectively. .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols. .. Libraries were sequenced in separate lanes on an Illumina HiSeq 2500 sequencer using a TruSeq SBS sequencing kit (version 3, Illumina) at the Genomics Core Facility, Purdue University.

Article Title: Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755
Article Snippet: Whole-genome sequencing of the NIES-3755 strain was performed using MiSeq sequencer (Illumina, San Diego, CA) and in silico finishing software as reported previously ( ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. 300 bp of each end of the libraries were sequenced on the MiSeq instrument with the MiSeq reagent kit v3 (600-cycles; Illumina).

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation
Article Snippet: The libraries were sequenced on the GS FLX+ instrument, yielding 309,976 shotgun reads and 222,244 paired-end reads. .. For MiSeq, an 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. The libraries were sequenced on the MiSeq instrument with the MiSeq reagent kit version 2 (500 cycles; Illumina), which yielded 668,946 paired-end reads and 539,927 mate-pair reads.

Article Title: Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium
Article Snippet: We obtained 259,087 fragment reads and 287,202, 308,517, and 318,239 paired-end reads with insert sizes of 8 kbp, 20 kbp, and 30 kbp, respectively. .. For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively. .. We obtained 2,201,763 paired-end reads, with an insert size of 800 bp.

Article Title: Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia
Article Snippet: Gene Set Enrichment Analysis (GSEA) of the 177 differentially expressed targets included testing against GO BP, Biocarata, Kegg, and Reactome gene sets. .. Total RNA was isolated from samples taken from bone marrow at diagnosis of leukemia, and again during remission. cDNA libraries were constructed for each sample using the TruSeq mRNA sample preparation kit (Illumina, San Diego CA). .. Poly-A containing mRNA from the total RNA is isolated using poly-T oligo-attached magnetic bead selection.

Article Title: m6A facilitates hippocampus-dependent learning and memory through Ythdf1
Article Snippet: Total RNA from WT littermate control and Ythdf1 -KO mouse (8–16 weeks, male) hippocampus was extracted using Trizol (Invitrogen) and isopropanol precipitation. mRNA extraction was performed by poly(A)+ RNA selection once using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen). .. The RNA libraries were prepared using Truseq stranded mRNA sample preparation kit (Illumina) according to manufacturer’s protocol. .. Adult male, 6~8-week old C57BL/6 mice were used (Charles River) and housed in a standard facility.

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: The resulting correlation was significant (p < 2E-16; adjusted R2 =0.2221) .. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina). .. Samples were individually indexed for pooling using a dual-index strategy.

Article Title: Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells
Article Snippet: Prior to sequencing, FAIRE DNA was analysed and quantified by running 1/25 of the FAIRE material on a High sensitivity DNA chip on a 2100 Bioanalyzer (Agilent, USA). .. 20 ng of FAIRE DNA was used for library preparation according to manufacturer’s instructions using the ChIP-seq sample preparation kit (Illumina). .. Single-read sequencing (36bp) was performed on a Genome Analyzer II (Illumina).

Next-Generation Sequencing:

Article Title: The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency
Article Snippet: Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina). .. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 200ng of total RNA using the TruSeq Stranded mRNA HT sample preparation kit (Illumina).

Produced:

Article Title: Draft Genome Sequence of Phomopsis longicolla Type Strain TWH P74, a Fungus Causing Phomopsis Seed Decay in Soybean
Article Snippet: A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols. .. A no-gel mate-pair library was generated with the Nextera mate-pair sample preparation kit (Illumina, San Diego, CA), and a paired-end library was made using the TruSeq DNA PCR-free sample preparation kit (Illumina, San Diego, CA) according to the manufacturer’s protocols.

In Silico:

Article Title: Complete Genome Sequence of Cyanobacterium Leptolyngbya sp. NIES-3755
Article Snippet: Whole-genome sequencing of the NIES-3755 strain was performed using MiSeq sequencer (Illumina, San Diego, CA) and in silico finishing software as reported previously ( ). .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation
Article Snippet: For MiSeq, an 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. For MiSeq, an 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of Rhodovulum sulfidophilum DSM 2351, an Extracellular Nucleic Acid-Producing Bacterium
Article Snippet: For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively. .. For MiSeq sequencing, a paired-end library and mate-pair libraries were constructed with the TruSeq DNA PCR-free sample preparation kit (Illumina) and the Nextera mate-pair sample preparation kit (Illumina), respectively.

Article Title: Complete Genome Sequence of the Bacteriochlorophyll b-Producing Photosynthetic Bacterium Blastochloris viridis
Article Snippet: An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively. .. An 800-bp paired-end library and an 8-kbp mate-pair library were prepared using the TruSeq DNA PCR-free sample preparation kit (Illumina) and Nextera mate-pair sample preparation kit (Illumina), respectively.

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    Illumina Inc nextera xt library preparation kit
    Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the <t>Nextera</t> XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.
    Nextera Xt Library Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nextera xt library preparation kit/product/Illumina Inc
    Average 99 stars, based on 165 article reviews
    Price from $9.99 to $1999.99
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    99
    Illumina Inc truseq rna library preparation kit v2
    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: <t>TruSeq</t> using 4 ng of <t>RNA;</t> Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients
    Truseq Rna Library Preparation Kit V2, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc nextera xt technology
    Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the <t>Nextera</t> XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.
    Nextera Xt Technology, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Concentration Assay, Generated, Software, Incubation, Magnetic Beads, DNA Purification, Fluorescence, Purification

    High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: High Throughput Screening Assay, Plasmid Preparation, Next-Generation Sequencing, Isolation, Sonication, Neutralization, Polymerase Chain Reaction, Purification, Concentration Assay

    Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Fluorescence, Concentration Assay, Purification, Standard Deviation

    Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Incubation, Purification, Concentration Assay, Sequencing

    Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Concentration Assay, Magnetic Beads, DNA Purification, Purification

    Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Article Snippet: 2.1 JMP® Pro 13 (SAS, UK) was used to generate custom designs for the optimisation of the library preparation of plasmid DNA samples for NGS, using the Nextera XT library preparation kit (FC-131-1096, Illumina Inc., USA).

    Techniques: Plasmid Preparation, Next-Generation Sequencing, Purification, Sequencing, Fluorescence, Concentration Assay, Picogreen Assay

    Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering of expression levels, based on the rank of the count of exon per million mapped reads (CPM). Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples.. Color scale: Spearman correlation coefficients

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques: Expressing

    Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Hierarchical clustering based on the rank of IP/input value. Dendrogram represents Spearman correlation coefficients between pairs of samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques:

    Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Descriptive characteristic of enrichment or depletion profiles as generated by the different library preparation kits. Genes which have at least 20 raw reads in the input samples and a ratio of IP/Input ≥2 or Input/IP ≥2 were used to generate the plots. a Total number of transcripts enriched or depleted. b Percentage of enriched or depleted transcripts grouped into different bins. X-axis: log2(IP/input), Y-axis: percentage of genes in each bin over whole population. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques: Generated

    Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Enrichment profiles and top 50 enriched transcripts. a Enrichment factor of transcripts are sorted in decreasing order based on log2 (IP/input). X-axis:transcripts, Y-axis:log2 value of enrichment (IP/Input). b Boxplot of top 50 enriched transcripts. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuGEN samples; Green and grey: TruSeq samples

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques:

    Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Descriptive characteristics of raw and mapped reads. a Total number of raw reads and number of reads mapped to the mouse genome (mm10, GRCm38.84). b Percentage of reads mapped to exonic, intronic and intergenic regions. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques:

    Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Distribution of normalized mean expression of the first (last) 100 bases of transcripts (in 5′- > 3′-orientation). X axis represents the 5′-3′ normalized position; Y axis represents normalized coverage. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA. Yellow and orange: SMTseq samples; Red: SMTer samples; Black: NEB samples; Blue: NuG samples; Green and grey: TruSeq samples. Solid: Input samples. Dotted: Ribo-IP samples

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques: Expressing

    Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Journal: BMC Genomics

    Article Title: A comparative analysis of library prep approaches for sequencing low input translatome samples

    doi: 10.1186/s12864-018-5066-2

    Figure Lengend Snippet: Venn diagrams of identified features in the different libraries. The features with CPM ≥ 1 in at least one out of 3 replicates were used to generate these plots. a and c represent input samples and b and d represent IP samples. Most transcripts were detected by all kits tested. However, a higher rate of agreement is seen between the NEB, TruSeq and SMART-Seq prepared samples. NEB: NEBNext® Ultra™, NuG: NuGEN Ovation®, SMTer: SMARTer® Stranded; Tru4: TruSeq using 4 ng of RNA; Tru70: TruSeq using 70 ng of RNA. SMTseq4: SMART-Seq® v4 using 4 ng of RNA; SMTseq0.25: SMART-Seq® v4 using 250 pg of RNA

    Article Snippet: We performed a hybrid library preparation by using Ovation® RNA-Seq System V2 (NuGEN, San Carlos, CA, USA) to synthesize cDNA and the TruSeq RNA Library Preparation Kit v2 to construct the sequencing library (Illumina, San Diego, CA, USA), consistent with the NuGEN manufacturer protocol (See Additional file : Table S1).

    Techniques:

    Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 2 for NGS plasmid DNA library preparation: Optimisation of fragment size and concentration. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time , plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were two response variables to be optimised: the peak fragment size (representing the size of most DNA fragments); and the peak relative fluorescence unit (RFU), indicative of concentration. A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that the tagmentation incubation time and DNA sample volume had a significant effect individually on the output variables. There was also a significant interaction between these two input variables. ( D ) The peak fragment size predicted by the DOE model correlated with the actual data with an R 2 value of 0.98, while the predicted peak RFU data correlated with the actual data with an R 2 value of 0.93, both indicative of a very good correlation. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp and maximum RFU), the optimised conditions suggested by the model are a DNA sample volume of 126 nl and a tagmentation incubation time of 12 min. ( F ) When the optimised conditions were tested on a multiwell plate of 96 samples, some samples had the desired peak fragment size (200–400 bp), however, many had larger fragment sizes than desired. Therefore, although the conditions optimised here are applicable to some plasmid DNA samples, further optimisation was required to establish the correct conditions for all plasmid DNA preparations.

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Concentration Assay, Generated, Software, Incubation, Magnetic Beads, DNA Purification, Fluorescence, Purification

    High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: High-throughput workflow for the preparation of plasmid DNA libraries for NGS. Plasmid DNA samples are isolated from bacteria cells using a high-throughput plasmid isolation method on the CyBio ® FeliX robot. All steps performed using the FeliX platform are highlighted with a grey outline. The isolated plasmid samples are tested for the presence of genomic DNA (gDNA), prior to library preparation, using the Labcyte Echo ® . All steps performed using the Labcyte Echo ® are highlighted with a shaded grey box. If samples are free from gDNA, they are diluted to 0.4 ng/μl in H 2 O. If gDNA is detected, the samples are sonicated prior to re-testing in the gDNA QC assay. Using reagents from the Nextera XT kit, a tagmentation reaction is performed on all samples under the optimised conditions, followed by neutralization of the reaction. Unique combinations of index primers are added to all samples via 12 PCR cycles, followed by magnetic bead purification of the PCR products. The concentration of the purified dsDNA is then determined using the PicoGreen ® reagent assay and the libraries are pooled to give a final concentration of 4–10 nM, in a minimum volume of 15 μl. The average fragment size of the pooled libraries is measured using the Fragment Analyzer before being sequenced on the Illumina ® MiSeq system.

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: High Throughput Screening Assay, Plasmid Preparation, Next-Generation Sequencing, Isolation, Sonication, Neutralization, Polymerase Chain Reaction, Purification, Concentration Assay

    Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 3 for NGS plasmid DNA library preparation: Optimising the reproducibility between different plasmid DNA preparations. ( A ) Two factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time and plasmid DNA sample volume. The evaluated range for each variable is shown. ( B ) There was a total of 11 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. There were three response variables: the peak fragment size (representing the size at which most DNA fragments are); the peak relative fluorescence unit (RFU); and the concentration of the sample, after magnetic bead purification. 8 plasmid DNA samples were prepared with the Nextera XT library preparation kit using the miniaturised method, according to each of the conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. The concentration of the purified samples was also determined using the PicoGreen ® dsDNA quantification assay. ( C ) The data were modelled according to the DOE design using JMP ® software. The effect summary shows that tagmentation incubation time and DNA sample volume each had a significant effect individually. There was also a significant interaction between the two variables. ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.48, 0.93 and 0.92 for the peak fragment size, peak RFU and concentration respectively. ( E ) The prediction profiler tool in the JMP ® software was used to visualise the data. When the desirability was maximised (peak fragment size of 200–400 bp, maximum RFU, a concentration of 0.5–5 ng/μl and minimised peak fragment size standard deviation), the optimised conditions suggested by the model are a DNA sample volume of 58.7 nl and a tagmentation incubation time of 12.5 min. ( F ) The Fragment Analyzer outputs for 8 samples, run with a 12.5 min tagmentation incubation time and 50 nl DNA, show that 7/8 of the samples have a peak fragment size of between 200 and 300 bp and an average concentration of 0.68 ng/μl (±0.33 SD). The undetected sample (sample 4) had a concentration below the limit of detection of the Fragment Analyzer (

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Fluorescence, Concentration Assay, Purification, Standard Deviation

    Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Next generation sequencing of plasmid DNA libraries, prepared using a miniaturised method with the Nextera XT library preparation kit. 96 plasmid DNA libraries were prepared for NGS with the Nextera XT library preparation kit, using optimised conditions (12.5 min incubation, 50 nl sample, 1.8x magnetic bead solution). ( A ) After purification, the samples were quantified in the PicoGreen ® dsDNA quantification assay. The data show that 75/92 samples have a concentration within the desired range (0.5–5 ng/μl), with an average concentration of 1.1 ng/μl. These samples were pooled, with a final concentration of 6.64 nM and run on the Fragment Analyzer ( B ) and ( C ). All fragments in the pooled libraries are of the desired size (200–400 bp). ( D ) The pooled library was sequenced on the Illumina ® MiSeq system (2 × 150 method). The mean sequence quality (Phred) scores are plotted for each sample. For all samples, the sequence quality (Phred) score was > 30 for more than 85% of the base pairs, indicating that all samples passed the QC criteria.

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Incubation, Purification, Concentration Assay, Sequencing

    Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Design of experiments (DOE) model number 1 for NGS plasmid DNA library preparation: Optimisation of the lower size limit of fragments. ( A ) Three factors were evaluated using a custom designed model generated with JMP ® software: tagmentation incubation time, plasmid DNA sample volume, and the concentration of magnetic beads used in the automated DNA purification method. The evaluated range for each variable is shown. ( B ) There was a total of 15 runs in random order from 5 whole plots. Each whole plot represents the same condition for the tagmentation incubation time and was performed in a separate plate. The response variable to be optimised was the lower size limit (the lowest size of DNA fragment detected after magnetic bead purification). A plasmid DNA sample was prepared with the Nextera XT library preparation kit using the miniaturised method, according to each set of conditions defined in the DOE model. After magnetic bead purification, samples were run neat on the Fragment Analyzer. ( C ) The data were modelled according to the DOE design, using JMP ® software. Both bead concentration and tagmentation incubation time has a significant effect on the lower size limit of the fragmented DNA (Log Worth > 2). ( D ) The data predicted by the DOE model correlated with the actual data with an R 2 value of 0.96, indicating a very good correlation. ( E ) Visualisation of the optimal lower size limit model using the prediction profiler tool in the JMP ® software, shows that the desired lower size limit of less than 300 bp is achieved with a tagmentation incubation time of > 7.5 min and a magnetic bead concentration of between 1.1–1.8x sample volume.

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: Next-Generation Sequencing, Plasmid Preparation, Generated, Software, Incubation, Concentration Assay, Magnetic Beads, DNA Purification, Purification

    Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Journal: Synthetic and Systems Biotechnology

    Article Title: Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    doi: 10.1016/j.synbio.2019.01.002

    Figure Lengend Snippet: Preparation of plasmid DNA libraries for NGS using miniaturised gDNA method. ( A ) Eight plasmid DNA samples were prepared according to Labcyte's miniaturised NGS gDNA library preparation method [ 2 ], using the Nextera XT kit. After purification, the samples were run on the Fragment Analyzer to determine the size of the fragments. ( B ) Plots of the Fragment Analyzer data show the peak fragment size for 7/8 of the samples is greater than 400 bp, which is the maximum limit required for sequencing. ( C ) Summary of the data shows variable relative fluorescence units (RFU) for the 8 samples. The concentration of each purified sample, as measured using the PicoGreen dsDNA assay, is within the range required (0.5–5 ng/μl).

    Article Snippet: Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system.

    Techniques: Plasmid Preparation, Next-Generation Sequencing, Purification, Sequencing, Fluorescence, Concentration Assay, Picogreen Assay