dna input  (New England Biolabs)


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    Name:
    NEBNext Ultra Ligation Module
    Description:
    NEBNext Ultra Ligation Module 96 rxns
    Catalog Number:
    E7445L
    Price:
    1195
    Category:
    DNA Template Preparation for PCR
    Size:
    96 rxns
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    Structured Review

    New England Biolabs dna input
    NEBNext Ultra Ligation Module
    NEBNext Ultra Ligation Module 96 rxns
    https://www.bioz.com/result/dna input/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna input - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples"

    Article Title: Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

    Journal: bioRxiv

    doi: 10.1101/094789

    Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.
    Figure Legend Snippet: Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.

    Techniques Used: Sequencing, DNA Extraction, Ethanol Precipitation, Sample Prep

    2) Product Images from "Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing"

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2757-4

    Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation
    Figure Legend Snippet: Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Techniques Used: Ligation

    3) Product Images from "Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples"

    Article Title: Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02483-16

    Timelines. We compared the method of Brown et al. with the results of this study obtained with Illumina MiSeq and MiniSeq and ONT MinION. We assumed that no step of the process can be initiated after 6 p.m. or before 8 a.m. The method of Brown et al. has a rapid extraction step but also a 20-h overnight enrichment step, resulting in a 50-h turnaround time. In our study, we did 27-h MiSeq runs (paired 150-bp reads), but since Mykrobe is k-mer based, a 16-h run (paired 75-bp reads) would give equivalent results; we therefore display that potential timeline here. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use, this would take 3 h. The 1.5-h orange rectangle on the MinION time lines includes both PCR and the 10-min sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 h. This image is intended to show comparable real-use time lines, and so the MiniSeq/MinION time lines are shown with 3-h extraction steps. MiniSeq enables a 16-h turnaround time by sequencing for only 7 h. R9 MinION also delivers sub-24-h results but requires one flow cell per sample. R9.4 MinION gives a 12.5-h turnaround time (6 h of sequencing with real-time [i.e., simultaneous] base calling when used on a single sample).
    Figure Legend Snippet: Timelines. We compared the method of Brown et al. with the results of this study obtained with Illumina MiSeq and MiniSeq and ONT MinION. We assumed that no step of the process can be initiated after 6 p.m. or before 8 a.m. The method of Brown et al. has a rapid extraction step but also a 20-h overnight enrichment step, resulting in a 50-h turnaround time. In our study, we did 27-h MiSeq runs (paired 150-bp reads), but since Mykrobe is k-mer based, a 16-h run (paired 75-bp reads) would give equivalent results; we therefore display that potential timeline here. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use, this would take 3 h. The 1.5-h orange rectangle on the MinION time lines includes both PCR and the 10-min sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 h. This image is intended to show comparable real-use time lines, and so the MiniSeq/MinION time lines are shown with 3-h extraction steps. MiniSeq enables a 16-h turnaround time by sequencing for only 7 h. R9 MinION also delivers sub-24-h results but requires one flow cell per sample. R9.4 MinION gives a 12.5-h turnaround time (6 h of sequencing with real-time [i.e., simultaneous] base calling when used on a single sample).

    Techniques Used: DNA Extraction, Ethanol Precipitation, Polymerase Chain Reaction, Sample Prep, Sequencing, Flow Cytometry

    4) Product Images from "Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing"

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2757-4

    Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation
    Figure Legend Snippet: Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Techniques Used: Ligation

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing
    Article Snippet: This allows the quantification of not only the overall yield, as normally done with qPCR, but also of the yield of some critical intermediate steps such as the adaptor ligation [ – ]. .. We present here the quantitative comparison of 9 kits: NEBNext and NEBNext Ultra from New England Biolabs, SureSelectXT from Agilent, Truseq Nano and Truseq DNA PCR-free from Illumina, Accel-NGS 1S and Accel-NGS 2S from Swift Biosciences, and KAPA Hyper and KAPA HyperPlus from KAPA Biosystems. .. All libraries were prepared using the same DNA sample (barcoded amplicons from phiX174 [ ]), and the different kits where compared in terms of overall and stepwise efficiencies, DNA loss, protocol length, flexibility and complexity.

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing
    Article Snippet: .. The fastest protocols are the NEBNext Ultra kit and the KAPA kits, particularly the KAPA HyperPlus, and up to certain extend the Illumina Truseq DNA PCR-free. .. Certain kits are specifically designed for low DNA input such as the NEBNext Ultra and Swift Accel-NGS 2S, while others such as the KAPA ones accept a wide range of DNA input from a 1 ng to 1 μg.

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq
    Article Snippet: An initial test amplification was used to determine the optimal cycle number for each library. .. For this, 1.25 μl library was amplified in 10 μl total volume with 0.4 μl each of the NEBNext Universal and any NEBNext Index primers with 5 μl NEBNext Ultra II Q5 PCR master mix. .. Cycling program: 98°C 30s then 18 cycles of (98°C 10 s, 65°C 75 s), 65°C 5 min. Test PCR was cleaned with 8 μl AMPure XP beads (Beckman A63881) and eluted with 2.5 μl 0.1x TE, of which 1 μl was examines on a Bioanalyser high sensitivity DNA chip (Agilent 5067-4626).

    Ligation:

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq
    Article Snippet: Library preparation: TrAEL-seq adaptor 2 was added using a modified NEBNext Ultra II DNA kit (NEB E7645S): 3.5 μl NEBNext Ultra II End Prep buffer, 1 μl 1 ng/μl sonicated salmon sperm DNA (this is used as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. .. After cooling, 1.25 μl 10 pM/μl TrAEL-seq adaptor 2, 0.5 μl NEBNext ligation enhancer and 15 μl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. ..

    Article Title: Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq
    Article Snippet: Library preparation: TrAEL-seq adaptor 2 was added using a modified NEBNext Ultra II DNA kit (NEB E7645S): 3.5 μl NEBNext Ultra II End Prep buffer, 1 μl 1 ng/μl sonicated salmon sperm DNA (this is used as a carrier), and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. .. After cooling, 1.25 μl 10 pM/μl TrAEL-seq adaptor 2, 0.5 μl NEBNext ligation enhancer, and 15 μl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. ..

    Incubation:

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq
    Article Snippet: Library preparation: TrAEL-seq adaptor 2 was added using a modified NEBNext Ultra II DNA kit (NEB E7645S): 3.5 μl NEBNext Ultra II End Prep buffer, 1 μl 1 ng/μl sonicated salmon sperm DNA (this is used as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. .. After cooling, 1.25 μl 10 pM/μl TrAEL-seq adaptor 2, 0.5 μl NEBNext ligation enhancer and 15 μl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. ..

    Article Title: Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq
    Article Snippet: Library preparation: TrAEL-seq adaptor 2 was added using a modified NEBNext Ultra II DNA kit (NEB E7645S): 3.5 μl NEBNext Ultra II End Prep buffer, 1 μl 1 ng/μl sonicated salmon sperm DNA (this is used as a carrier), and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. .. After cooling, 1.25 μl 10 pM/μl TrAEL-seq adaptor 2, 0.5 μl NEBNext ligation enhancer, and 15 μl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. ..

    Amplification:

    Article Title: Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq
    Article Snippet: An initial test amplification was used to determine the optimal cycle number for each library. .. For this, 1.25 μl library was amplified in 10 μl total volume with 0.4 μl each of the NEBNext Universal and any NEBNext Index primers with 5 μl NEBNext Ultra II Q5 PCR master mix. .. Cycling program: 98°C 30s then 18 cycles of (98°C 10 s, 65°C 75 s), 65°C 5 min. Test PCR was cleaned with 8 μl AMPure XP beads (Beckman A63881) and eluted with 2.5 μl 0.1x TE, of which 1 μl was examines on a Bioanalyser high sensitivity DNA chip (Agilent 5067-4626).

    Selection:

    Article Title: Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq
    Article Snippet: .. Both TruSeq Nano and NEBNext Ultra methods have very similar work flows, and rely on SPRi bead-based size selection. .. The Illumina protocol adds index sequences during adaptor ligation, whilst the NEBNext Ultra protocol adds indexes to adaptor tagged fragments during the PCR enrichment steps, but these differences do not significantly impact on workflow.

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    New England Biolabs dna input
    Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the <t>ONT</t> MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The <t>DNA</t> extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.
    Dna Input, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna input/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna input - by Bioz Stars, 2021-07
    86/100 stars
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    99
    New England Biolabs nebuilder hifi assembly input dna fragments
    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: <t>DNA</t> ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using <t>NEBuilder</t> <t>HiFi</t> (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.
    Nebuilder Hifi Assembly Input Dna Fragments, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi assembly input dna fragments/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    97
    New England Biolabs dna inputs
    Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq <t>DNA</t> Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length <t>Illumina</t> TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.
    Dna Inputs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.

    Journal: bioRxiv

    Article Title: Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

    doi: 10.1101/094789

    Figure Lengend Snippet: Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.

    Article Snippet: These samples, along with pure BCG DNA, were prepared following ONTs PCR-based protocol for low-input libraries (DP006_revB_14Aug2015), using modified primers supplied by ONT, a 20 ng DNA input into the PCR reaction, and LongAmp Taq 2X Master Mix (New England Biolabs, USA).

    Techniques: Sequencing, DNA Extraction, Ethanol Precipitation, Sample Prep

    Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Journal: BMC Genomics

    Article Title: Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing

    doi: 10.1186/s12864-016-2757-4

    Figure Lengend Snippet: Example of yield measurements obtained with the NEBNext kit when comparing the amount of DNA at each step versus the initial DNA input (500 ng). Blue bars correspond to values measured using the PhiX specific primers and reflect the DNA loss at each step mainly due to pipetting and bead clean up. The orange bar corresponds to the value measured with the adaptor specific primers and reflects the amount of DNA in the sample bearing adaptor after the ligation step in comparison with the initial DNA input. The green bar corresponds to the value measured with the P5/P7 primers and reflects the amount of sequencable DNA in the sample at the end of the library preparation

    Article Snippet: Certain kits are specifically designed for low DNA input such as the NEBNext Ultra and Swift Accel-NGS 2S, while others such as the KAPA ones accept a wide range of DNA input from a 1 ng to 1 μg.

    Techniques: Ligation

    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Article Snippet: NEBuilder HiFi assembly input DNA fragments were modified as before in triplicate reactions (PCR overhang filling by Q5 polymerase).

    Techniques: Polymerase Chain Reaction, Clone Assay, Negative Control, Ligation, Plasmid Preparation

    Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Article Snippet: NEBuilder HiFi assembly input DNA fragments were modified as before in triplicate reactions (PCR overhang filling by Q5 polymerase).

    Techniques: Polymerase Chain Reaction, Clone Assay, Ligation, Amplification, Plasmid Preparation

    Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Article Snippet: NEBuilder HiFi assembly input DNA fragments were modified as before in triplicate reactions (PCR overhang filling by Q5 polymerase).

    Techniques: Clone Assay, Plasmid Preparation, Inverse PCR, Activity Assay, Transformation Assay

    Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Article Snippet: NEBuilder HiFi assembly input DNA fragments were modified as before in triplicate reactions (PCR overhang filling by Q5 polymerase).

    Techniques: Ligation, Functional Assay, Construct, Standard Deviation, Two Tailed Test, Isolation, Transformation Assay, Clone Assay, Polymerase Chain Reaction

    Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Journal: PLoS ONE

    Article Title: Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

    doi: 10.1371/journal.pone.0096727

    Figure Lengend Snippet: Overview of the different methods used for adapter addition to antibody variable heavy chain amplicon libraries. All methods required the reverse transcription of antibody mRNA into cDNA (step 1), which served as template for the following IgG gene-specific amplification by PCR. (A) The ligation method required a pre-amplified library as starting material, with a 3′ A-overhang added by the Taq DNA Polymerase (step 2). The stem-loop adapters containing a 5′ T-overhang were then attached in an enzymatic ligation reaction and cleaved in order to create a double-stranded form (step 3) that served as template for a final amplification step (step 4) in which the full-length Illumina TruSeq universal and index adapter sequences were incorporated into the library. (B) The direct addition method combined antibody library amplification and sequencing adapter addition into one PCR step (step 2) by attaching the Illumina adapter sequences 5′ of the gene-specific primers used for library preparation. (C) The primer extension method incorporated a GC-rich overhang into the library in PCR1 (step 2). This resulted in uniformly high amplification in a second PCR by using primers specific for the GC-rich overhang and containing the full-length Illumina sequencing adapters (step 3). UTR: untranslated region, L: leader sequence, V: variable region, C: constant region, RT: reverse transcription, fw: forward, rv: reverse, x: barcode/index allowing multiplexed sequencing runs.

    Article Snippet: It should be noted that kits are now available that offer the ligation addition of Illumina adapters with starting DNA inputs as little as 5 ng (NEBNext Ultra, NEB).

    Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing