Structured Review

Illumina Inc illumina barcodes
Workflow for IGH sequencing from bulk gDNA. ( a ) Starting from PBMCs, bone marrow aspirate, or formalin-fixed paraffin-embedded samples, gDNA is extracted from bulk populations. ( b ) Next, IGH gene rearrangements are amplified from gDNA using primer cocktails in FR1 and JH along with <t>Illumina</t> adapters. V = variable, D = diversity, and J = joining genes. ( c ) Amplicons from this first round of PCR are purified using AMPure beads and ( d ) subjected to second-round amplification using primers that include sample <t>barcodes</t> (see primers in Subheading 2.1 for DNA sequence information). ( e ) Sequencing libraries are subjected to further purification, size selection, quality control, and pooling prior to loading onto the sequencer
Illumina Barcodes, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 98/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/illumina barcodes/product/Illumina Inc
Average 98 stars, based on 40 article reviews
Price from $9.99 to $1999.99
illumina barcodes - by Bioz Stars, 2022-10
98/100 stars

Images

1) Product Images from "Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distribution: A Method by the AIRR Community"

Article Title: Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distribution: A Method by the AIRR Community

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-0716-2115-8_18

Workflow for IGH sequencing from bulk gDNA. ( a ) Starting from PBMCs, bone marrow aspirate, or formalin-fixed paraffin-embedded samples, gDNA is extracted from bulk populations. ( b ) Next, IGH gene rearrangements are amplified from gDNA using primer cocktails in FR1 and JH along with Illumina adapters. V = variable, D = diversity, and J = joining genes. ( c ) Amplicons from this first round of PCR are purified using AMPure beads and ( d ) subjected to second-round amplification using primers that include sample barcodes (see primers in Subheading 2.1 for DNA sequence information). ( e ) Sequencing libraries are subjected to further purification, size selection, quality control, and pooling prior to loading onto the sequencer
Figure Legend Snippet: Workflow for IGH sequencing from bulk gDNA. ( a ) Starting from PBMCs, bone marrow aspirate, or formalin-fixed paraffin-embedded samples, gDNA is extracted from bulk populations. ( b ) Next, IGH gene rearrangements are amplified from gDNA using primer cocktails in FR1 and JH along with Illumina adapters. V = variable, D = diversity, and J = joining genes. ( c ) Amplicons from this first round of PCR are purified using AMPure beads and ( d ) subjected to second-round amplification using primers that include sample barcodes (see primers in Subheading 2.1 for DNA sequence information). ( e ) Sequencing libraries are subjected to further purification, size selection, quality control, and pooling prior to loading onto the sequencer

Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction, Purification, Selection

2) Product Images from "MinION barcodes: biodiversity discovery and identification by everyone, for everyone"

Article Title: MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Journal: bioRxiv

doi: 10.1101/2021.03.09.434692

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)

Techniques Used:

Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).

Techniques Used:

3) Product Images from "Full-Length Spatial Transcriptomics Reveals the Unexplored Isoform Diversity of the Myocardium Post-MI"

Article Title: Full-Length Spatial Transcriptomics Reveals the Unexplored Isoform Diversity of the Myocardium Post-MI

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2022.912572

Defining morphological regions after MI. (A) Dot plot showing the expresson of selected markers associated with the expresson of CM = cardiomyocytes, EC = endothelial cells, MFB = myofibroblasts, IM = immune cells, or with fibrosis and inflammation, based on the short-read Illumina data. (B) Annotation of mouse heart regions after MI via short-read clustering, transfered to the Nanopore data. Scatter plot in spatial coordinates of the anatomical regions (left) and UMAP representation of the Nanopore data using the region annotation from short-read clustering (right). Colors in the spatial scatter plot are matching those of the UMAP. (C) Neighbors enrichment analysis in one heart axis section. The heatmap shows the enrichment score on spatial proximity between the different anatomical regions. Spots belonging to two different regions that are close together will have a high score, and vice-versa. (D) Cluster co-occurrence in spatial dimensions in one heart axis section. Line plot showing the conditional probability of observing a given region conditioned on the presence of the infarct region, computed across increasing radii size around each spots. Distance units are given in pixels of the Visium source image. (E) Barplot showing the frequency distribution of the number of isoforms per gene, either stemming from the assembly, or found in the final data after quality control filtering. The median length of transcripts is indicated in each bar for each category. (F) Average number of isoform per gene detected among markers of each morphological region. Significance was measured using a Mann-Whitney U-test (*** =
Figure Legend Snippet: Defining morphological regions after MI. (A) Dot plot showing the expresson of selected markers associated with the expresson of CM = cardiomyocytes, EC = endothelial cells, MFB = myofibroblasts, IM = immune cells, or with fibrosis and inflammation, based on the short-read Illumina data. (B) Annotation of mouse heart regions after MI via short-read clustering, transfered to the Nanopore data. Scatter plot in spatial coordinates of the anatomical regions (left) and UMAP representation of the Nanopore data using the region annotation from short-read clustering (right). Colors in the spatial scatter plot are matching those of the UMAP. (C) Neighbors enrichment analysis in one heart axis section. The heatmap shows the enrichment score on spatial proximity between the different anatomical regions. Spots belonging to two different regions that are close together will have a high score, and vice-versa. (D) Cluster co-occurrence in spatial dimensions in one heart axis section. Line plot showing the conditional probability of observing a given region conditioned on the presence of the infarct region, computed across increasing radii size around each spots. Distance units are given in pixels of the Visium source image. (E) Barplot showing the frequency distribution of the number of isoforms per gene, either stemming from the assembly, or found in the final data after quality control filtering. The median length of transcripts is indicated in each bar for each category. (F) Average number of isoform per gene detected among markers of each morphological region. Significance was measured using a Mann-Whitney U-test (*** =

Techniques Used: MANN-WHITNEY

scNaST methodology. (A) Schematic of the scNaST workflow using a hybrid sequencing approach on Nanopore and Illumina platforms to assign spatial barcodes to long-read sequencing. (B) Nanopore sequencing saturation showing the number of splice sites detected at various levels of subsampling. A curve that reaches a plateau before getting to 100% data suggest that all known junctions in the library have been detected. The curve shows the mean ± SE of four samples. (C) Normalized transcript coverage for Nanopore and Illumina. The curves show the mean ± SE of four samples. (D) Reads assigned by scNapBar at each step of the workflow. The bars show the mean ± SE of four samples.
Figure Legend Snippet: scNaST methodology. (A) Schematic of the scNaST workflow using a hybrid sequencing approach on Nanopore and Illumina platforms to assign spatial barcodes to long-read sequencing. (B) Nanopore sequencing saturation showing the number of splice sites detected at various levels of subsampling. A curve that reaches a plateau before getting to 100% data suggest that all known junctions in the library have been detected. The curve shows the mean ± SE of four samples. (C) Normalized transcript coverage for Nanopore and Illumina. The curves show the mean ± SE of four samples. (D) Reads assigned by scNapBar at each step of the workflow. The bars show the mean ± SE of four samples.

Techniques Used: Sequencing, Nanopore Sequencing

4) Product Images from "7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli"

Article Title: 7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkac148

Schematic representation of the in vivo mutagenesis assay with NextGen sequencing. X is either A (adenine) or oxo-ϵA at the lesion site. The colored box to the left of the interrogated site symbolizes the lesion-specific trinucleotide barcode; the box to the right of the interrogated site represents a second barcode introduced during Illumina library preparation (indexing barcode), corresponding to each sample.
Figure Legend Snippet: Schematic representation of the in vivo mutagenesis assay with NextGen sequencing. X is either A (adenine) or oxo-ϵA at the lesion site. The colored box to the left of the interrogated site symbolizes the lesion-specific trinucleotide barcode; the box to the right of the interrogated site represents a second barcode introduced during Illumina library preparation (indexing barcode), corresponding to each sample.

Techniques Used: In Vivo, Mutagenesis, Sequencing

5) Product Images from "MinION barcodes: biodiversity discovery and identification by everyone, for everyone"

Article Title: MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Journal: bioRxiv

doi: 10.1101/2021.03.09.434692

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)

Techniques Used:

Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).

Techniques Used:

6) Product Images from "ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone"

Article Title: ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

Journal: BMC Biology

doi: 10.1186/s12915-021-01141-x

Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)

Techniques Used:

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)

Techniques Used:

7) Product Images from "Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction"

Article Title: Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction

Journal: Microbial Genomics

doi: 10.1099/mgen.0.000744

Barcode distribution and G+C bias of Hackflex barcode v1 libraries. Unique barcode distribution across 96 Hackflex libraries constructed from barcodes v1 (left) and their G+C bias: the relation between barcode counts obtained for each entire barcode (i.e. F5 +i5+N5 or F7 +i7+N7), and its G+C content (i5, R =−0.17, P =0.1; i7, R =0.03, P =0.77) (right).
Figure Legend Snippet: Barcode distribution and G+C bias of Hackflex barcode v1 libraries. Unique barcode distribution across 96 Hackflex libraries constructed from barcodes v1 (left) and their G+C bias: the relation between barcode counts obtained for each entire barcode (i.e. F5 +i5+N5 or F7 +i7+N7), and its G+C content (i5, R =−0.17, P =0.1; i7, R =0.03, P =0.77) (right).

Techniques Used: Construct

8) Product Images from "ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone"

Article Title: ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

Journal: BMC Biology

doi: 10.1186/s12915-021-01141-x

Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)

Techniques Used:

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)

Techniques Used:

9) Product Images from "Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis"

Article Title: Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis

Journal: Genome Biology

doi: 10.1186/gb-2011-12-9-r93

Quality assessment of the Illumina sequence data . (a) Number of reads with barcodes that passed Illumina filtering and aligned to the reference templates using Bowtie from individually indexed libraries ( n = 12). Range, 641 k to 978 k reads; mean ± standard deviation, 809 k ± 107 k. (b) Percentage of total (unaligned) reads that fall into a mean Phred quality interval. Note > 80% of the reads have mean Phred quality scores ≥25. (c) Nucleotide content as a function of sequencing cycles ( n = 47). Note that the nucleotide proportions closely match the expected proportions as determined from the templates.
Figure Legend Snippet: Quality assessment of the Illumina sequence data . (a) Number of reads with barcodes that passed Illumina filtering and aligned to the reference templates using Bowtie from individually indexed libraries ( n = 12). Range, 641 k to 978 k reads; mean ± standard deviation, 809 k ± 107 k. (b) Percentage of total (unaligned) reads that fall into a mean Phred quality interval. Note > 80% of the reads have mean Phred quality scores ≥25. (c) Nucleotide content as a function of sequencing cycles ( n = 47). Note that the nucleotide proportions closely match the expected proportions as determined from the templates.

Techniques Used: Sequencing, Standard Deviation

10) Product Images from "A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling"

Article Title: A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling

Journal: BMC Genomics

doi: 10.1186/s12864-015-2194-9

Experimental design. ( a ) Design of single and dual-index sequencing strategy and schematic describing the 3 amplicon designs: Fusion Primer Design (A) is a one step PCR which uses a single 12-nt error-correcting Golay index sequence (blue) allowing a high multiplexing capability. Tag tailed design (B) is a 2-step PCR which uses a universal primer for the first step and a dual index barcoded primer set in the second step. Standard Illumina Nextera 8-nt index sequences were used (pink Index 5; blue Index 7). The Pac Bio Ligate Adapters design (C): Two harpin adapters (grey) were ligated to a barcoded template (BF forward barcode; BR reverse barcode) to allow multiplexing. ( b ) Platform Specific Amplicon Libraries: Illumina paired-end sequencing (1,2) generates 2 sequencing reads (R1 and R2) per each cluster and can have single (Standard/Golay) or dual indexes (I5, I7). Ion Torrent and 454 (3) have a single read for each bead with a single index (MID). Pacific Bioscience generate a single circular read for each molecule (SMRT bell) and can have one (BF or BR) or two indexes. The starting point and direction of sequencing reads are indicated by a solid blue line and arrows, respectively. In the case of Fusion Primer Design custom sequencing primer were used
Figure Legend Snippet: Experimental design. ( a ) Design of single and dual-index sequencing strategy and schematic describing the 3 amplicon designs: Fusion Primer Design (A) is a one step PCR which uses a single 12-nt error-correcting Golay index sequence (blue) allowing a high multiplexing capability. Tag tailed design (B) is a 2-step PCR which uses a universal primer for the first step and a dual index barcoded primer set in the second step. Standard Illumina Nextera 8-nt index sequences were used (pink Index 5; blue Index 7). The Pac Bio Ligate Adapters design (C): Two harpin adapters (grey) were ligated to a barcoded template (BF forward barcode; BR reverse barcode) to allow multiplexing. ( b ) Platform Specific Amplicon Libraries: Illumina paired-end sequencing (1,2) generates 2 sequencing reads (R1 and R2) per each cluster and can have single (Standard/Golay) or dual indexes (I5, I7). Ion Torrent and 454 (3) have a single read for each bead with a single index (MID). Pacific Bioscience generate a single circular read for each molecule (SMRT bell) and can have one (BF or BR) or two indexes. The starting point and direction of sequencing reads are indicated by a solid blue line and arrows, respectively. In the case of Fusion Primer Design custom sequencing primer were used

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Multiplexing

11) Product Images from "Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A"

Article Title: Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2021.662782

Depiction of strategy for memory B cell sorting and BCR transcript sequencing. (A) PBMCs from a healthy donor were labeled with either SpA KK or control HSA-tetramers. (B) The memory B cell compartment positive for the SpA KK tetramer was identified and sorted. (C) The sorted population was collected, with single cell analysis that incorporates unique barcodes and high-throughput sequencing. Reads of BCR transcripts were analyzed for antibody gene usage and repertoire characteristics.
Figure Legend Snippet: Depiction of strategy for memory B cell sorting and BCR transcript sequencing. (A) PBMCs from a healthy donor were labeled with either SpA KK or control HSA-tetramers. (B) The memory B cell compartment positive for the SpA KK tetramer was identified and sorted. (C) The sorted population was collected, with single cell analysis that incorporates unique barcodes and high-throughput sequencing. Reads of BCR transcripts were analyzed for antibody gene usage and repertoire characteristics.

Techniques Used: FACS, Sequencing, Labeling, Single-cell Analysis, Next-Generation Sequencing

12) Product Images from "Hackflex: low cost Illumina Nextera Flex sequencing library construction"

Article Title: Hackflex: low cost Illumina Nextera Flex sequencing library construction

Journal: bioRxiv

doi: 10.1101/779215

Barcode distribution and GC bias of Hackflex barcode v1 libraries. Unique barcode distribution across 96 Hackflex libraries constructed from barcodes v1(left) and their GC bias: the relation between barcode counts obtained for each entire barcode (i.e.: F5+i5+N5 or F7+i7+N7), and its GC content (i5 R =-0.17 p=0.1; i7 R =0.03 p=0.77) (right).
Figure Legend Snippet: Barcode distribution and GC bias of Hackflex barcode v1 libraries. Unique barcode distribution across 96 Hackflex libraries constructed from barcodes v1(left) and their GC bias: the relation between barcode counts obtained for each entire barcode (i.e.: F5+i5+N5 or F7+i7+N7), and its GC content (i5 R =-0.17 p=0.1; i7 R =0.03 p=0.77) (right).

Techniques Used: Construct

13) Product Images from "Single-Nucleotide-Resolution Computing and Memory in Living Cells"

Article Title: Single-Nucleotide-Resolution Computing and Memory in Living Cells

Journal: bioRxiv

doi: 10.1101/263657

Incorporating memory and logic in living cells by DOMINO. A) Schematic representation of DOMINO operators. DOMINO operators are enabled by a DNA read-write head that performs efficient and precise manipulation of genomic DNA with single-nucleotide resolution. In this device, nCas9 (READ module), along with cytidine deaminase (CDA, WRITE module) and uracil DNA glycosylase ( ugi , WRITE enhancer) domains are addressed to a desired genomic loci using gRNA with a complementary seed region (READ address). Localization of the CDA write module to the target results in the deamination of cytidine (dC) residues in the vicinity of the 5’-end of the gRNA (WRITE address) and their conversion to dU residues, which are then preferentially repaired by the cellular machinery to dT (or dG to dA mutation if the negative strand of DNA is targeted by gRNA). By placing the DNA read-write module and the gRNA under the control of inducible signals, DNA writing for DOMINO operators can be tuned and controlled by external cues. Here, we schematize the basic DOMINO operator as an AND gate since it requires the expression of both the DNA read-write head (i.e., CDA-nCas9-ugi controlled by the “operational signal”) as well as the gRNA (regulated by “Input 1”) with a downstream feedback delay operator (to illustrate the unidirectional and memory aspect of the operator). DOMINO operators can be layered to a wide variety of memory and logic functions. Bold nucleotides on the target show the location of NGG PAM sequence. Targeted nucleotides are underlined. B) Order-independent AND gate enabled by DOMINO where the output is ON only when both inputs have been present with any possible order. Induction of the circuit with either of the two inducers (IPTG or Ara), results in editing of the target and transition to an intermediate state (states S1 or S2, respectively). Induction of the circuit with both gRNAs results in generation of the doubly edited DNA sequence (state S3), which is designated as ON state. C) Dynamics of allele frequencies obtained by Illumina High-Throughput Sequencing (HTS) for the circuit shown in (B). E. coli cells were exposed to different inducer combinations for four days with serial dilution after each 24 hours. Error bars indicate standard deviation of three biological replicates. D) Position-specific mutant allele frequencies for the last time point (96 h) of the experiment shown in (C) estimated from Sanger sequencing analysis by Sequalizer (see Supplementary Materials). This data demonstrates the expected outcomes of AND gate behavior at the population level. The x-axis shows dC to dT or dG to dA mutations in the specified positions. For example, the G18A mutation means a dG to dA mutation in position 18 of the target sequence. Small boxes along the x-axis show the induction patterns and duration of induction used in each experiment. For example, the induction pattern of the last sample set ([IA][IA][IA][IA]) means that the samples were induced with aTc + IPTG + Ara for four days with dilutions every 24 hours. Error bars indicate standard deviation of three biological replicates. E) The output of DOMINO operators, which is in the form of DNA mutations, can be converted to a gRNA by flanking the target DNA sequence with a desired promoter and gRNA handle. This allows DOMINO operators to be linked to other DOMINO operators or host regulatory networks. To demonstrate this concept, we designed an order-independent DOMINO AND gate with a target sequence flanked by a constitutive promoter and a modified gRNA handle. The modified gRNA handle harbored a dA to dG mutation in a position that was not essential for gRNA function ( Briner et al., 2014 ). This modification (shown by an asterisk) was required to generate an NGG PAM motif for binding of one of the input gRNAs. Upon induction by both inducers, the input gRNAs edit the Specificity-Determining Sequence (SDS) of the output gRNA. The doubly edited output gRNA can then bind to the GFP ORF and repress it via CRISPRi in E. coli . In this example, AND logic is realized on the target DNA register (i.e., the output gRNA) while NAND logic is achieved on the output GFP reporter. Error bars indicate standard deviation for three biological replicates.
Figure Legend Snippet: Incorporating memory and logic in living cells by DOMINO. A) Schematic representation of DOMINO operators. DOMINO operators are enabled by a DNA read-write head that performs efficient and precise manipulation of genomic DNA with single-nucleotide resolution. In this device, nCas9 (READ module), along with cytidine deaminase (CDA, WRITE module) and uracil DNA glycosylase ( ugi , WRITE enhancer) domains are addressed to a desired genomic loci using gRNA with a complementary seed region (READ address). Localization of the CDA write module to the target results in the deamination of cytidine (dC) residues in the vicinity of the 5’-end of the gRNA (WRITE address) and their conversion to dU residues, which are then preferentially repaired by the cellular machinery to dT (or dG to dA mutation if the negative strand of DNA is targeted by gRNA). By placing the DNA read-write module and the gRNA under the control of inducible signals, DNA writing for DOMINO operators can be tuned and controlled by external cues. Here, we schematize the basic DOMINO operator as an AND gate since it requires the expression of both the DNA read-write head (i.e., CDA-nCas9-ugi controlled by the “operational signal”) as well as the gRNA (regulated by “Input 1”) with a downstream feedback delay operator (to illustrate the unidirectional and memory aspect of the operator). DOMINO operators can be layered to a wide variety of memory and logic functions. Bold nucleotides on the target show the location of NGG PAM sequence. Targeted nucleotides are underlined. B) Order-independent AND gate enabled by DOMINO where the output is ON only when both inputs have been present with any possible order. Induction of the circuit with either of the two inducers (IPTG or Ara), results in editing of the target and transition to an intermediate state (states S1 or S2, respectively). Induction of the circuit with both gRNAs results in generation of the doubly edited DNA sequence (state S3), which is designated as ON state. C) Dynamics of allele frequencies obtained by Illumina High-Throughput Sequencing (HTS) for the circuit shown in (B). E. coli cells were exposed to different inducer combinations for four days with serial dilution after each 24 hours. Error bars indicate standard deviation of three biological replicates. D) Position-specific mutant allele frequencies for the last time point (96 h) of the experiment shown in (C) estimated from Sanger sequencing analysis by Sequalizer (see Supplementary Materials). This data demonstrates the expected outcomes of AND gate behavior at the population level. The x-axis shows dC to dT or dG to dA mutations in the specified positions. For example, the G18A mutation means a dG to dA mutation in position 18 of the target sequence. Small boxes along the x-axis show the induction patterns and duration of induction used in each experiment. For example, the induction pattern of the last sample set ([IA][IA][IA][IA]) means that the samples were induced with aTc + IPTG + Ara for four days with dilutions every 24 hours. Error bars indicate standard deviation of three biological replicates. E) The output of DOMINO operators, which is in the form of DNA mutations, can be converted to a gRNA by flanking the target DNA sequence with a desired promoter and gRNA handle. This allows DOMINO operators to be linked to other DOMINO operators or host regulatory networks. To demonstrate this concept, we designed an order-independent DOMINO AND gate with a target sequence flanked by a constitutive promoter and a modified gRNA handle. The modified gRNA handle harbored a dA to dG mutation in a position that was not essential for gRNA function ( Briner et al., 2014 ). This modification (shown by an asterisk) was required to generate an NGG PAM motif for binding of one of the input gRNAs. Upon induction by both inducers, the input gRNAs edit the Specificity-Determining Sequence (SDS) of the output gRNA. The doubly edited output gRNA can then bind to the GFP ORF and repress it via CRISPRi in E. coli . In this example, AND logic is realized on the target DNA register (i.e., the output gRNA) while NAND logic is achieved on the output GFP reporter. Error bars indicate standard deviation for three biological replicates.

Techniques Used: Mutagenesis, Expressing, Sequencing, Acetylene Reduction Assay, Next-Generation Sequencing, Serial Dilution, Standard Deviation, Modification, Binding Assay

14) Product Images from "Mutation bias reflects natural selection in Arabidopsis thaliana"

Article Title: Mutation bias reflects natural selection in Arabidopsis thaliana

Journal: Nature

doi: 10.1038/s41586-021-04269-6

Sequencing depth, mappability, and false positives do not explain observed biases in distributions of natural polymorphisms or observed mutations used to predict mutation probabilities. a , Sequencing depth around transcription start (TSS) and termination (TTS) sites in one randomly chosen mutation accumulation line. b , Mappability around TSS TTS site calculated with GenMap 47 . c , Rates of false positive SNP and InDel calls around TSS and TTS determined from 1,000 iterations of simulated Illumina reads. d , Simulation of effect of selection on gene bodies. Selection could take the form of mutations being dominant lethal or through somatic competition of mutations with small selection coefficients. 0 = 0%, 0.01 = 1%, 0.1 = 10%, 0.2 = 20%, 0.3 = 30% of gene body mutations removed by purifying selection. 30% is estimated to be the approximate upper bound of constrained sites in gene bodies 34 . e – i , Resequencing of 10 siblings of one MA line from ref. 12 . e , Overview of experimental design for testing the effect of sequencing depth on calling somatic mutations. f , Filtered heterozygous variants (SNVs and InDels) called in sibling #5 sequenced at ~600x depth overlap more with variants called from sibling #5 at ~60x sequencing depth than with other siblings at ~60 sequencing depth. The boxplots show the distribution of 20 iterations of sampling equal numbers of heterozygous variants (to account for differences in total number of variants called in different siblings) for each sibling sequenced at ~60x and compared to sibling 5 sequenced at ~600x. Boxplots show median with maxima and minima reflecting interquartile range (IQR), whiskers = 1.5 * IQR (n = 20 iterations). g , Frequency distribution of unfiltered heterozygous variants called in 10 siblings sequenced at ~60x depth each. Note that because these siblings are descendants of 25 generations of self-fertilization, the number of true heterozygous (inherited segregating) calls is expected to be very small compared to heterozygous variants that are chimeric somatic mutations. h , Average mappability of variants detected in different numbers of siblings out of the 10 sequenced siblings. i , Variants called independently in one sibling or, less so, in two to four siblings show signatures of mutation bias. In contrast, variants called in five or more siblings (which should more likely be false positives due to cryptic duplications or regions with poor mappability) do not show a biased distribution around TSS and TSS, with overall distribution similar to known false positives.
Figure Legend Snippet: Sequencing depth, mappability, and false positives do not explain observed biases in distributions of natural polymorphisms or observed mutations used to predict mutation probabilities. a , Sequencing depth around transcription start (TSS) and termination (TTS) sites in one randomly chosen mutation accumulation line. b , Mappability around TSS TTS site calculated with GenMap 47 . c , Rates of false positive SNP and InDel calls around TSS and TTS determined from 1,000 iterations of simulated Illumina reads. d , Simulation of effect of selection on gene bodies. Selection could take the form of mutations being dominant lethal or through somatic competition of mutations with small selection coefficients. 0 = 0%, 0.01 = 1%, 0.1 = 10%, 0.2 = 20%, 0.3 = 30% of gene body mutations removed by purifying selection. 30% is estimated to be the approximate upper bound of constrained sites in gene bodies 34 . e – i , Resequencing of 10 siblings of one MA line from ref. 12 . e , Overview of experimental design for testing the effect of sequencing depth on calling somatic mutations. f , Filtered heterozygous variants (SNVs and InDels) called in sibling #5 sequenced at ~600x depth overlap more with variants called from sibling #5 at ~60x sequencing depth than with other siblings at ~60 sequencing depth. The boxplots show the distribution of 20 iterations of sampling equal numbers of heterozygous variants (to account for differences in total number of variants called in different siblings) for each sibling sequenced at ~60x and compared to sibling 5 sequenced at ~600x. Boxplots show median with maxima and minima reflecting interquartile range (IQR), whiskers = 1.5 * IQR (n = 20 iterations). g , Frequency distribution of unfiltered heterozygous variants called in 10 siblings sequenced at ~60x depth each. Note that because these siblings are descendants of 25 generations of self-fertilization, the number of true heterozygous (inherited segregating) calls is expected to be very small compared to heterozygous variants that are chimeric somatic mutations. h , Average mappability of variants detected in different numbers of siblings out of the 10 sequenced siblings. i , Variants called independently in one sibling or, less so, in two to four siblings show signatures of mutation bias. In contrast, variants called in five or more siblings (which should more likely be false positives due to cryptic duplications or regions with poor mappability) do not show a biased distribution around TSS and TSS, with overall distribution similar to known false positives.

Techniques Used: Sequencing, Mutagenesis, Selection, Sampling

15) Product Images from "AutoRELACS: automated generation and analysis of ultra-parallel ChIP-seq"

Article Title: AutoRELACS: automated generation and analysis of ultra-parallel ChIP-seq

Journal: Scientific Reports

doi: 10.1038/s41598-020-69443-8

AutoRELACS workflow ensures comprehensive integration of RELACS barcodes. ( a ) Overview of AutoRELACS protocol. ( 1-M) Nuclei of formaldehyde-fixed cells are extracted manually using adjusted ultrasound 14 . The nuclear envelope is permeabilized, and the chromatin digested in situ using a 4-cutter restriction enzyme (RE). (2-A) Digested chromatin from each sample is automatically barcoded. Upon completion, the liquid handler pools all barcoded samples into a unique tube (Biomek i7 program: “RELACS_Barcoding”). (3-M) Pooled samples are collected by the user and nuclei are lysed using focused sonication. (4-A) The barcoded chromatin is aliquoted according to the number of required immunoprecipitation (IP) reactions into corresponding ChIP reaction mixes. The ChIP reactions are carried out overnight in parallel at room temperature on the Biomek i7 workstation. Upon completion, the ChIP-ped chromatin is sequestrated using beads and automatically washed 4 times at increasing stringency conditions and finally eluted in the elution buffer (Biomek program: “RELACS_ChIP_Elution”). (5-A) Subsequently, the eluted chromatin is decrosslinked and the DNA is purified. DNA is amplified via PCR using primers carrying Illumina dual indexes. Optionally, the liquid handler performs multiple rounds of purification and size selection using Ampure XP beads (Biomek program: “RELACS_Decrosslink_FinalLibraries”). A: Automated; M: Manual. Cells images in step1 were made by Freepick from www.flaticon.com . The image was created using Adobe Inc. (2020). Adobe Illustrator . Retrieved from https://adobe.com/products/illustrator . (6-A) Libraries are sequenced on Illumina’s sequencing devices. Upon completion of the sequencing run, bcl2 files are automatically converted to fastq format and input into the fully automated ChIP-seq workflow available as part of the snakePipes suite 13 . SnakePipes’ ChIP-seq workflow performs demultiplexing of reads on RELACS custom barcodes, quality controls, mapping and filtering of duplicate reads using unique molecular identifiers (UMI), and further downstream analysis like generation of input-normalized coverage tracks and peak calling. ( b ) Distribution of RELACS barcodes in two independent input chromatin pools. 60 barcodes are integrated into the digested chromatin of two independent batches of S2 cells. Sequencing of the input chromatin pool for replicate 1 (upper panel) and replicate 2 (lower panel), reveals the percentage of input reads for each barcode used (y-axis). The ideal uniform distribution (100/60) is represented as a dotted line. The shaded gray area shows one standard deviation from the mean of the observed distribution.
Figure Legend Snippet: AutoRELACS workflow ensures comprehensive integration of RELACS barcodes. ( a ) Overview of AutoRELACS protocol. ( 1-M) Nuclei of formaldehyde-fixed cells are extracted manually using adjusted ultrasound 14 . The nuclear envelope is permeabilized, and the chromatin digested in situ using a 4-cutter restriction enzyme (RE). (2-A) Digested chromatin from each sample is automatically barcoded. Upon completion, the liquid handler pools all barcoded samples into a unique tube (Biomek i7 program: “RELACS_Barcoding”). (3-M) Pooled samples are collected by the user and nuclei are lysed using focused sonication. (4-A) The barcoded chromatin is aliquoted according to the number of required immunoprecipitation (IP) reactions into corresponding ChIP reaction mixes. The ChIP reactions are carried out overnight in parallel at room temperature on the Biomek i7 workstation. Upon completion, the ChIP-ped chromatin is sequestrated using beads and automatically washed 4 times at increasing stringency conditions and finally eluted in the elution buffer (Biomek program: “RELACS_ChIP_Elution”). (5-A) Subsequently, the eluted chromatin is decrosslinked and the DNA is purified. DNA is amplified via PCR using primers carrying Illumina dual indexes. Optionally, the liquid handler performs multiple rounds of purification and size selection using Ampure XP beads (Biomek program: “RELACS_Decrosslink_FinalLibraries”). A: Automated; M: Manual. Cells images in step1 were made by Freepick from www.flaticon.com . The image was created using Adobe Inc. (2020). Adobe Illustrator . Retrieved from https://adobe.com/products/illustrator . (6-A) Libraries are sequenced on Illumina’s sequencing devices. Upon completion of the sequencing run, bcl2 files are automatically converted to fastq format and input into the fully automated ChIP-seq workflow available as part of the snakePipes suite 13 . SnakePipes’ ChIP-seq workflow performs demultiplexing of reads on RELACS custom barcodes, quality controls, mapping and filtering of duplicate reads using unique molecular identifiers (UMI), and further downstream analysis like generation of input-normalized coverage tracks and peak calling. ( b ) Distribution of RELACS barcodes in two independent input chromatin pools. 60 barcodes are integrated into the digested chromatin of two independent batches of S2 cells. Sequencing of the input chromatin pool for replicate 1 (upper panel) and replicate 2 (lower panel), reveals the percentage of input reads for each barcode used (y-axis). The ideal uniform distribution (100/60) is represented as a dotted line. The shaded gray area shows one standard deviation from the mean of the observed distribution.

Techniques Used: In Situ, Sonication, Immunoprecipitation, Chromatin Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Selection, Sequencing, Standard Deviation

16) Product Images from "ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone"

Article Title: ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

Journal: BMC Biology

doi: 10.1186/s12915-021-01141-x

Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)

Techniques Used:

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)

Techniques Used:

17) Product Images from "A Drying-Rewetting Cycle Imposes More Important Shifts on Soil Microbial Communities than Does Reduced Precipitation"

Article Title: A Drying-Rewetting Cycle Imposes More Important Shifts on Soil Microbial Communities than Does Reduced Precipitation

Journal: mSystems

doi: 10.1128/msystems.00247-22

Bacterial and archaeal (A) and fungal (B) community compositions at the phylum level (mean relative abundance > 1%). The mean relative abundance was calculated based on Illumina amplicon sequencing of the 16S rRNA gene for bacteria and archaea and the ITS region for fungi.
Figure Legend Snippet: Bacterial and archaeal (A) and fungal (B) community compositions at the phylum level (mean relative abundance > 1%). The mean relative abundance was calculated based on Illumina amplicon sequencing of the 16S rRNA gene for bacteria and archaea and the ITS region for fungi.

Techniques Used: Amplification, Sequencing

18) Product Images from "MinION barcodes: biodiversity discovery and identification by everyone, for everyone"

Article Title: MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Journal: bioRxiv

doi: 10.1101/2021.03.09.434692

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84-97%). Percentage of barcodes recovered is relative to the final estimate based on all data.

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x-axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents number of demultiplexed reads over time (plotted against Z-axis)

Techniques Used:

Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5-200X coverage shows that there are very minor gains to barcode quality after 25-50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red).

Techniques Used:

19) Product Images from "Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools"

Article Title: Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools

Journal: Cold Spring Harbor Perspectives in Biology

doi: 10.1101/cshperspect.a032375

RIG-seq for high-throughput retrotransposon profiling. ( A ) RIG assay. The intron donor plasmid pLNRK-RIG carries the Ll.LtrB intron (red) flanked by exons (gray) under a nisin-inducible promoter (P nisA ). The intron is interrupted by a retromobility indicator gene (RIG) with the kan R gene inserted in the anti-transcriptional orientation to the intron and carrying its own promoter (P kan ). The kan R gene is interrupted by a self-splicing group I intron (gpI) in the same orientation as Ll.LtrB (red arrow). ( B ) Reconstitution of the kan R gene with formation of a characteristic splice junction (SJ) sequence and kanamycin resistance is possible only through reverse transcription of an RNA intermediate that lost the group I intron during retrotransposition. Wavy line to the left is chromosomal DNA, whereas plasmid to the right is the RIG donor. ( C ) RIG-seq amplification scheme. High-throughput targeted sequencing of insertion loci was based on the generation of the SJ sequence of the kan R gene during retrotransposition. After ligation of Illumina-specific adapters, P5A and P7A, two tandem polymerase chain reactions (PCRs) were used to amplify flanks of retrotransposition events in Illumina libraries: A specific SJ primer (SJ_oligo) with the P7_oligo is used in the first PCR; then a library-specific primer, the chimeric oligo, carrying library-specific indices, is used with the P7_oligo in the second PCR. Pseq is the sequencing primer. ( D ) Density of intron-insertions in part of the Lactococcus lactis .)
Figure Legend Snippet: RIG-seq for high-throughput retrotransposon profiling. ( A ) RIG assay. The intron donor plasmid pLNRK-RIG carries the Ll.LtrB intron (red) flanked by exons (gray) under a nisin-inducible promoter (P nisA ). The intron is interrupted by a retromobility indicator gene (RIG) with the kan R gene inserted in the anti-transcriptional orientation to the intron and carrying its own promoter (P kan ). The kan R gene is interrupted by a self-splicing group I intron (gpI) in the same orientation as Ll.LtrB (red arrow). ( B ) Reconstitution of the kan R gene with formation of a characteristic splice junction (SJ) sequence and kanamycin resistance is possible only through reverse transcription of an RNA intermediate that lost the group I intron during retrotransposition. Wavy line to the left is chromosomal DNA, whereas plasmid to the right is the RIG donor. ( C ) RIG-seq amplification scheme. High-throughput targeted sequencing of insertion loci was based on the generation of the SJ sequence of the kan R gene during retrotransposition. After ligation of Illumina-specific adapters, P5A and P7A, two tandem polymerase chain reactions (PCRs) were used to amplify flanks of retrotransposition events in Illumina libraries: A specific SJ primer (SJ_oligo) with the P7_oligo is used in the first PCR; then a library-specific primer, the chimeric oligo, carrying library-specific indices, is used with the P7_oligo in the second PCR. Pseq is the sequencing primer. ( D ) Density of intron-insertions in part of the Lactococcus lactis .)

Techniques Used: High Throughput Screening Assay, Plasmid Preparation, Sequencing, Amplification, Ligation, Polymerase Chain Reaction

Three methods of using thermostable group II intron reverse transcriptase (TGIRT) enzymes for RNA-seq library construction (TGIRT-seq). ( A ) TGIRT-seq of eukaryotic mRNAs using an anchored oligo(dT) 42 primer for initiation of cDNA synthesis. The primer of sufficient length to stably anneal to mRNA poly(A) tails at high temperature is extended by TGIRTs at 60°C to synthesize cDNA copies of the mRNAs. After second-strand synthesis, dsDNAs are fragmented and RNA-seq adapters are added by conventional methods, such as the transposon-based Illumina Nextera method. ( B ). After reverse transcription, cDNAs are purified on a denaturing polyacrylamide gel to select specific size classes, circularized with CircLigase (Epicentre), and minimally polymerase chain reaction (PCR) amplified with primers that add capture sites and barcodes for Illumina sequencing. ( C ) TGIRT Total RNA-seq method for construction of comprehensive RNA-seq libraries without size selection. The TGIRT initiates from an initial template-primer substrate similar to that in panel B but containing only an Illumina R2 adapter sequence. After reverse transcription, cDNAs are cleaned up by using a MinElute column to remove unincorporated adapters and a second oligonucleotide containing the reverse complement of an Illumina R1 adapter is ligated to the 3′ end of the cDNA using thermostable 5′ AppDNA/RNA ligase (New England Biolabs). After an additional MinElute cleanup, the cDNAs with adapters on both ends are amplified by PCR with primers that add Illumina capture sites and barcodes. Before sequencing, the libraries are cleaned up using Ampure beads to remove adapter dimers (not shown).
Figure Legend Snippet: Three methods of using thermostable group II intron reverse transcriptase (TGIRT) enzymes for RNA-seq library construction (TGIRT-seq). ( A ) TGIRT-seq of eukaryotic mRNAs using an anchored oligo(dT) 42 primer for initiation of cDNA synthesis. The primer of sufficient length to stably anneal to mRNA poly(A) tails at high temperature is extended by TGIRTs at 60°C to synthesize cDNA copies of the mRNAs. After second-strand synthesis, dsDNAs are fragmented and RNA-seq adapters are added by conventional methods, such as the transposon-based Illumina Nextera method. ( B ). After reverse transcription, cDNAs are purified on a denaturing polyacrylamide gel to select specific size classes, circularized with CircLigase (Epicentre), and minimally polymerase chain reaction (PCR) amplified with primers that add capture sites and barcodes for Illumina sequencing. ( C ) TGIRT Total RNA-seq method for construction of comprehensive RNA-seq libraries without size selection. The TGIRT initiates from an initial template-primer substrate similar to that in panel B but containing only an Illumina R2 adapter sequence. After reverse transcription, cDNAs are cleaned up by using a MinElute column to remove unincorporated adapters and a second oligonucleotide containing the reverse complement of an Illumina R1 adapter is ligated to the 3′ end of the cDNA using thermostable 5′ AppDNA/RNA ligase (New England Biolabs). After an additional MinElute cleanup, the cDNAs with adapters on both ends are amplified by PCR with primers that add Illumina capture sites and barcodes. Before sequencing, the libraries are cleaned up using Ampure beads to remove adapter dimers (not shown).

Techniques Used: RNA Sequencing Assay, Stable Transfection, Purification, Polymerase Chain Reaction, Amplification, Sequencing, Selection

20) Product Images from "ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone"

Article Title: ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

Journal: BMC Biology

doi: 10.1186/s12915-021-01141-x

Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)
Figure Legend Snippet: Relationship between barcode quality and coverage. Subsetting the data to 5–200X coverage shows that there are very minor gains to barcode quality after 25–50X coverage. (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1 N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red)

Techniques Used:

Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data
Figure Legend Snippet: Relationship between barcoding success and number of raw reads for six amplicon pools (191-9932 specimens; barcoding success rates 84–97%). Percentage of barcodes recovered is relative to the final estimate based on all data

Techniques Used: Amplification

Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)
Figure Legend Snippet: Rapid recovery of accurate MinION barcodes over time (in hours, x -axis) (filtered barcodes: dark green = barcodes passing all 4 QC criteria, light green = one ambiguous base; lighter green = more than 1N, no barcode = white with pattern, 1 mismatch = orange, > 1 mismatch = red). The solid black line represents the number of barcodes available for comparison. White dotted line represents the amount of raw reads collected over time, blue represents the number of demultiplexed reads over time (plotted against Z -axis)

Techniques Used:

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Illumina Inc barcodes
    Flowchart for generating MinION <t>barcodes</t> from experimental set-up to final barcodes. The novel steps introduced in this study are highlighted in green and the scripts available in miniBarcoder for analyses are further indicated.
    Barcodes, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/barcodes/product/Illumina Inc
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    barcodes - by Bioz Stars, 2022-10
    88/100 stars
      Buy from Supplier

    90
    Illumina Inc barcode
    Comparative schematic of small RNA barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the <t>barcode</t> is incorporated in the 5' adapter. In the TruSeq method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.
    Barcode, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/barcode/product/Illumina Inc
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    barcode - by Bioz Stars, 2022-10
    90/100 stars
      Buy from Supplier

    88
    Illumina Inc amplicon sequencing
    Non-metric multidimensional scaling (NMDS) based on Bray-Curtis similarities of nematode community structures from field plots replanted five times with apple or virgin control plots planted one time with apple, analyzed by high-throughput 18S rDNA <t>amplicon</t> sequencing from total nematode community DNA. PERMANOVA revealed a significant difference in the overall nematode community structure between ARD and control soils ( P
    Amplicon Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplicon sequencing/product/Illumina Inc
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    amplicon sequencing - by Bioz Stars, 2022-10
    88/100 stars
      Buy from Supplier

    92
    illumina inc cell barcode assignment
    Sicelore and ScNapBar CPU time comparison. ( A ) ScNapBar CPU time depends on the number of whitelist barcodes (allowing an edit distance of > 2 and and offset of up to 4 bp between adapter and <t>barcode).</t> Gray area represents the standard deviation for 10 runs. ( B ) Comparison of ScNapBar and Sicelore CPU times. Benchmark was measured using one million barcode sequences and 2052 barcodes in the whitelist.
    Cell Barcode Assignment, supplied by illumina inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell barcode assignment/product/illumina inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cell barcode assignment - by Bioz Stars, 2022-10
    92/100 stars
      Buy from Supplier

    Image Search Results


    Flowchart for generating MinION barcodes from experimental set-up to final barcodes. The novel steps introduced in this study are highlighted in green and the scripts available in miniBarcoder for analyses are further indicated.

    Journal: bioRxiv

    Article Title: Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing

    doi: 10.1101/622365

    Figure Lengend Snippet: Flowchart for generating MinION barcodes from experimental set-up to final barcodes. The novel steps introduced in this study are highlighted in green and the scripts available in miniBarcoder for analyses are further indicated.

    Article Snippet: Indeed, even the initial estimates of barcodes (“MAFFT” & “RACON”) have very high accuracy ( > 99.5%) when compared to Illumina data, while the accuracy of consolidated barcodes is even higher ( > 99.9%).

    Techniques:

    Ambiguities in MAFFT+AA (Purple), RACON+AA (Yellow) and Consolidated barcodes (Green) with varying namino parameters (1,2 and 3). One outlier value for Racon+3AA barcode was excluded from the plot. The plot shows that the consolidated barcodes have few ambiguities remaining.

    Journal: bioRxiv

    Article Title: Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing

    doi: 10.1101/622365

    Figure Lengend Snippet: Ambiguities in MAFFT+AA (Purple), RACON+AA (Yellow) and Consolidated barcodes (Green) with varying namino parameters (1,2 and 3). One outlier value for Racon+3AA barcode was excluded from the plot. The plot shows that the consolidated barcodes have few ambiguities remaining.

    Article Snippet: Indeed, even the initial estimates of barcodes (“MAFFT” & “RACON”) have very high accuracy ( > 99.5%) when compared to Illumina data, while the accuracy of consolidated barcodes is even higher ( > 99.9%).

    Techniques:

    Effect of re-pooling on coverage of barcodes for both sets of specimens. Barcodes with coverage

    Journal: bioRxiv

    Article Title: Rapid, large-scale species discovery in hyperdiverse taxa using 1D MinION sequencing

    doi: 10.1101/622365

    Figure Lengend Snippet: Effect of re-pooling on coverage of barcodes for both sets of specimens. Barcodes with coverage

    Article Snippet: Indeed, even the initial estimates of barcodes (“MAFFT” & “RACON”) have very high accuracy ( > 99.5%) when compared to Illumina data, while the accuracy of consolidated barcodes is even higher ( > 99.9%).

    Techniques:

    Comparative schematic of small RNA barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the barcode is incorporated in the 5' adapter. In the TruSeq method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.

    Journal: PLoS ONE

    Article Title: Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing

    doi: 10.1371/journal.pone.0026969

    Figure Lengend Snippet: Comparative schematic of small RNA barcoding methods. The three methods start with ligation of a 3' and 5' RNA adapter to generate a substrate for RT-PCR. In the pre-PCR barcoding method, the barcode is incorporated in the 5' adapter. In the TruSeq method, the barcode is incorporated in one of the RT-PCR primers. In the PALM barcoding method, the amplified RT-PCR product is A-tailed and ligated to a T-tailed barcoded adapter.

    Article Snippet: The adapters used in the protocol were modified to include a barcode and to allow for Illumina index sequencing with the Illumina multiplexing index read sequencing primer.

    Techniques: Ligation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    Non-metric multidimensional scaling (NMDS) based on Bray-Curtis similarities of nematode community structures from field plots replanted five times with apple or virgin control plots planted one time with apple, analyzed by high-throughput 18S rDNA amplicon sequencing from total nematode community DNA. PERMANOVA revealed a significant difference in the overall nematode community structure between ARD and control soils ( P

    Journal: Frontiers in Plant Science

    Article Title: Free-Living Nematodes Together With Associated Microbes Play an Essential Role in Apple Replant Disease

    doi: 10.3389/fpls.2018.01666

    Figure Lengend Snippet: Non-metric multidimensional scaling (NMDS) based on Bray-Curtis similarities of nematode community structures from field plots replanted five times with apple or virgin control plots planted one time with apple, analyzed by high-throughput 18S rDNA amplicon sequencing from total nematode community DNA. PERMANOVA revealed a significant difference in the overall nematode community structure between ARD and control soils ( P

    Article Snippet: Barcoded amplicon sequencing of the 18S rRNA genes was done by 2 × 250 bp paired-end high-throughput sequencing on an Illumina MiSeq platform (Illumina, San Diego, CA, United States).

    Techniques: High Throughput Screening Assay, Amplification, Sequencing

    Sicelore and ScNapBar CPU time comparison. ( A ) ScNapBar CPU time depends on the number of whitelist barcodes (allowing an edit distance of > 2 and and offset of up to 4 bp between adapter and barcode). Gray area represents the standard deviation for 10 runs. ( B ) Comparison of ScNapBar and Sicelore CPU times. Benchmark was measured using one million barcode sequences and 2052 barcodes in the whitelist.

    Journal: RNA

    Article Title: Single-cell transcriptome sequencing on the Nanopore platform with ScNapBar

    doi: 10.1261/rna.078154.120

    Figure Lengend Snippet: Sicelore and ScNapBar CPU time comparison. ( A ) ScNapBar CPU time depends on the number of whitelist barcodes (allowing an edit distance of > 2 and and offset of up to 4 bp between adapter and barcode). Gray area represents the standard deviation for 10 runs. ( B ) Comparison of ScNapBar and Sicelore CPU times. Benchmark was measured using one million barcode sequences and 2052 barcodes in the whitelist.

    Article Snippet: We performed an in silico benchmark of cell barcode assignment when both, cell barcode and UMI, are found in the Nanopore read.

    Techniques: Standard Deviation

    Combined single-cell Illumina and Nanopore sequencing strategy. GFP+/− cells are pooled and sequenced on the Illumina and Nanopore platform. The Nanopore platform generates long cDNA sequencing reads that are used in barcode calling and estimating read error parameters. The Illumina data are used to estimate the total number of cells in sequencing and the represented cell barcodes. The simulated data are then used to parameterize a Bayesian model of barcode alignment features to discriminate correct versus false barcode assignments. This model is then used on the real data to assign cell barcodes to Nanopore reads. The GFP label and known NMD transcripts can be used to validate this assignment.

    Journal: RNA

    Article Title: Single-cell transcriptome sequencing on the Nanopore platform with ScNapBar

    doi: 10.1261/rna.078154.120

    Figure Lengend Snippet: Combined single-cell Illumina and Nanopore sequencing strategy. GFP+/− cells are pooled and sequenced on the Illumina and Nanopore platform. The Nanopore platform generates long cDNA sequencing reads that are used in barcode calling and estimating read error parameters. The Illumina data are used to estimate the total number of cells in sequencing and the represented cell barcodes. The simulated data are then used to parameterize a Bayesian model of barcode alignment features to discriminate correct versus false barcode assignments. This model is then used on the real data to assign cell barcodes to Nanopore reads. The GFP label and known NMD transcripts can be used to validate this assignment.

    Article Snippet: We performed an in silico benchmark of cell barcode assignment when both, cell barcode and UMI, are found in the Nanopore read.

    Techniques: Nanopore Sequencing, Sequencing

    Number of the Nanopore reads identified by ScNapBar and Sicelore at each processing step. We inspected each processing step on real data (low lllumina saturation of 11.3%). The first two steps are identical for both workflows. Total Reads: Number of input reads, aligned to genome: Number of reads aligned to genome. The next three steps are workflow-specific: Aligned to adapter: Number of reads with identified adapter sequence, aligned to barcode: Number of reads with aligned barcode sequence, Assigned to barcode: Number of predictions by each workflow. The last step is a validation of the previous assignment step after additional Illumina sequencing, which increases the Illumina saturation to 52%, and using UMI matches, see main text.

    Journal: RNA

    Article Title: Single-cell transcriptome sequencing on the Nanopore platform with ScNapBar

    doi: 10.1261/rna.078154.120

    Figure Lengend Snippet: Number of the Nanopore reads identified by ScNapBar and Sicelore at each processing step. We inspected each processing step on real data (low lllumina saturation of 11.3%). The first two steps are identical for both workflows. Total Reads: Number of input reads, aligned to genome: Number of reads aligned to genome. The next three steps are workflow-specific: Aligned to adapter: Number of reads with identified adapter sequence, aligned to barcode: Number of reads with aligned barcode sequence, Assigned to barcode: Number of predictions by each workflow. The last step is a validation of the previous assignment step after additional Illumina sequencing, which increases the Illumina saturation to 52%, and using UMI matches, see main text.

    Article Snippet: We performed an in silico benchmark of cell barcode assignment when both, cell barcode and UMI, are found in the Nanopore read.

    Techniques: Sequencing

    Sensitivity and specificity of ScNapBar and Sicelore on 100 Illumina libraries with different levels of saturation. ( A ) Barcode assignment with UMI matches. ( B ) Barcode assignment without UMI matches (ScNapBar score > 50). ( C ) Benchmark of the specificity and sensitivity of the Illumina library with 100% saturation. We compared the barcode assignments with ScNapBar score > 1–99, and the assignments from Sicelore with UMI support are roughly equivalent to the ScNapBar score > 90.

    Journal: RNA

    Article Title: Single-cell transcriptome sequencing on the Nanopore platform with ScNapBar

    doi: 10.1261/rna.078154.120

    Figure Lengend Snippet: Sensitivity and specificity of ScNapBar and Sicelore on 100 Illumina libraries with different levels of saturation. ( A ) Barcode assignment with UMI matches. ( B ) Barcode assignment without UMI matches (ScNapBar score > 50). ( C ) Benchmark of the specificity and sensitivity of the Illumina library with 100% saturation. We compared the barcode assignments with ScNapBar score > 1–99, and the assignments from Sicelore with UMI support are roughly equivalent to the ScNapBar score > 90.

    Article Snippet: We performed an in silico benchmark of cell barcode assignment when both, cell barcode and UMI, are found in the Nanopore read.

    Techniques: