Structured Review

Illumina Inc miseq system
Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion <t>S5</t> system ( 1a ), and using manual identification via the BlueFuse Multi on the <t>Miseq</t> system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
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1) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.
Figure Legend Snippet: Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.

Techniques Used:

2) Product Images from "PANDAA-monium: Intentional violations of conventional qPCR design enables rapid, HIV-1 subtype-independent drug resistance SNP detection"

Article Title: PANDAA-monium: Intentional violations of conventional qPCR design enables rapid, HIV-1 subtype-independent drug resistance SNP detection

Journal: bioRxiv

doi: 10.1101/795054

Validation of PANDAA performance. PANDAA was performed with differentially labeled TaqMan probes to discriminate wild-type DNA (VIC-labeled [green]) from the K103N DRM (FAM-labeled [red]). ( A ) Using either 100% wild-type DNA or 100% mutant 014 template DNA at 10 5 copies per reaction, non-specific DRM signal (red squares) can clearly be differentiated from the specific wild-type signal (green circles) in wild-type only reactions. Similarly, the non-specific wild-type signal (green squares) can be distinguished from the specific K103 DRM signal (red circles). ( B ) PANDAA was performed on 10-fold dilutions of a 1:1 mixture of 10 5 to 10 total DNA copies; thus, wild-type (green circles) and mutant DNA (red circles) were present at 50% of those quantities. ( C ) Mixed populations of wild-type (green) to mutant DNA (red), representing 10% K103N DRM at 10 5 total copies of DNA. ( D ) Negative control using human genomic DNA. Results are representative of a minimum of six replicates of each dilution series. The x-axis represents the number of qPCR cycles and the y-axis represents the log normalized fluorescence. ( E ) Correlation of PANDAA with NGS. The proportions of K65, K103N, Y181C, and M184VI mutations were quantified by Illumina MiSeq NGS. Pearson’s correlation coefficient showed a strong agreement between the DRM proportions quantified by PANDAA and those quantified by NGS (r = 0.9837; 95% CI: 0.9759–0.9890; P
Figure Legend Snippet: Validation of PANDAA performance. PANDAA was performed with differentially labeled TaqMan probes to discriminate wild-type DNA (VIC-labeled [green]) from the K103N DRM (FAM-labeled [red]). ( A ) Using either 100% wild-type DNA or 100% mutant 014 template DNA at 10 5 copies per reaction, non-specific DRM signal (red squares) can clearly be differentiated from the specific wild-type signal (green circles) in wild-type only reactions. Similarly, the non-specific wild-type signal (green squares) can be distinguished from the specific K103 DRM signal (red circles). ( B ) PANDAA was performed on 10-fold dilutions of a 1:1 mixture of 10 5 to 10 total DNA copies; thus, wild-type (green circles) and mutant DNA (red circles) were present at 50% of those quantities. ( C ) Mixed populations of wild-type (green) to mutant DNA (red), representing 10% K103N DRM at 10 5 total copies of DNA. ( D ) Negative control using human genomic DNA. Results are representative of a minimum of six replicates of each dilution series. The x-axis represents the number of qPCR cycles and the y-axis represents the log normalized fluorescence. ( E ) Correlation of PANDAA with NGS. The proportions of K65, K103N, Y181C, and M184VI mutations were quantified by Illumina MiSeq NGS. Pearson’s correlation coefficient showed a strong agreement between the DRM proportions quantified by PANDAA and those quantified by NGS (r = 0.9837; 95% CI: 0.9759–0.9890; P

Techniques Used: Labeling, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction, Fluorescence, Next-Generation Sequencing

3) Product Images from "Dysbiosis-Associated Polyposis of the Colon—Cap Polyposis"

Article Title: Dysbiosis-Associated Polyposis of the Colon—Cap Polyposis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00918

Fecal microbiota analysis in a patient with cap polyposis. (A) Stool samples were obtained from a patient with cap polyposis prior to, 1 week and 6 months post-antibiotic treatment. DNA samples extracted from the stool specimens were subjected to polymerase chain reaction for the amplification of the 16S ribosomal RNA (16S rRNA) V3 and V4 regions. We performed 16S rRNA sequencing using the MiSeq system. The relative abundance of different bacterial taxa at the genus level in each sample has been shown. (B) Comparative analysis of the taxonomic composition of the fecal microbial community at the genus level. Relative abundance of the genera has been shown as a percentage.
Figure Legend Snippet: Fecal microbiota analysis in a patient with cap polyposis. (A) Stool samples were obtained from a patient with cap polyposis prior to, 1 week and 6 months post-antibiotic treatment. DNA samples extracted from the stool specimens were subjected to polymerase chain reaction for the amplification of the 16S ribosomal RNA (16S rRNA) V3 and V4 regions. We performed 16S rRNA sequencing using the MiSeq system. The relative abundance of different bacterial taxa at the genus level in each sample has been shown. (B) Comparative analysis of the taxonomic composition of the fecal microbial community at the genus level. Relative abundance of the genera has been shown as a percentage.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

4) Product Images from "Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria"

Article Title: Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00786

Ploidy and replication manner of the chromosomes of C. aponinum PCC 10605 and Geminocystis sp. NIES-3708. Exponentially growing C. aponinum (chromosome approximately 4.1 Mbp), Geminocystis sp. (chromosome approximately 3.9 Mbp), and S. elongatus (chromosome approximately 2.7 Mbp; 3–6 copies per cell; Zheng and O’Shea, 2017 ) ( Supplementary Figure S2 ) were fixed and stained with SYBR Green and then examined using flow cytometry. (A) Images of SYBR Green-stained cells. Images were acquired using differential interference contrast microscopy (top) and fluorescence microscopy (bottom). (B) Distribution of DNA levels per cell and cell volumes of exponentially growing cultures of C. aponinum (blue), Geminocystis sp. (red), and S . elongatus (black). (C) Depths of the high-throughput genomic DNA sequence reads at their respective chromosomal regions. Genomic DNA was extracted from the exponentially growing cells ( Supplementary Figure S2 ) and analyzed using an Illumina MiSeq System. Plots of 1-kb (left) and 100-kb windows (right). The number of reads (divided by the number of total reads) of the growing (replicating) cells normalized by that of the stationary phase (non-replicating) cells at each genomic position is shown. The asterisk in the profile of S. elongatus indicates a ∼50-kb genomic deletion in our wild type strain which has little effect on replication and cellular growth ( Watanabe et al., 2012 ).
Figure Legend Snippet: Ploidy and replication manner of the chromosomes of C. aponinum PCC 10605 and Geminocystis sp. NIES-3708. Exponentially growing C. aponinum (chromosome approximately 4.1 Mbp), Geminocystis sp. (chromosome approximately 3.9 Mbp), and S. elongatus (chromosome approximately 2.7 Mbp; 3–6 copies per cell; Zheng and O’Shea, 2017 ) ( Supplementary Figure S2 ) were fixed and stained with SYBR Green and then examined using flow cytometry. (A) Images of SYBR Green-stained cells. Images were acquired using differential interference contrast microscopy (top) and fluorescence microscopy (bottom). (B) Distribution of DNA levels per cell and cell volumes of exponentially growing cultures of C. aponinum (blue), Geminocystis sp. (red), and S . elongatus (black). (C) Depths of the high-throughput genomic DNA sequence reads at their respective chromosomal regions. Genomic DNA was extracted from the exponentially growing cells ( Supplementary Figure S2 ) and analyzed using an Illumina MiSeq System. Plots of 1-kb (left) and 100-kb windows (right). The number of reads (divided by the number of total reads) of the growing (replicating) cells normalized by that of the stationary phase (non-replicating) cells at each genomic position is shown. The asterisk in the profile of S. elongatus indicates a ∼50-kb genomic deletion in our wild type strain which has little effect on replication and cellular growth ( Watanabe et al., 2012 ).

Techniques Used: Periodic Counter-current Chromatography, Staining, SYBR Green Assay, Flow Cytometry, Microscopy, Fluorescence, High Throughput Screening Assay, Sequencing

Chromosomal replication origins of Synechococcus sp. PCC 7002 WT and dnaA disruptants. Depth of the high-throughput genomic DNA reads at respective chromosomal regions in exponentially growing WT and Δ dnaA cells. Genomic DNA was extracted from cells 24 h after inoculation ( Figure 4B ) and analyzed using an Illumina MiSeq System. The number of reads (divided by the number of total reads) of the growing (replicating) cells was normalized by that of the stationary phase (non-replicating) cells at each genomic position. 1-kb window (left) and 100-kb window (right) of WT and Δ dnaA clones.
Figure Legend Snippet: Chromosomal replication origins of Synechococcus sp. PCC 7002 WT and dnaA disruptants. Depth of the high-throughput genomic DNA reads at respective chromosomal regions in exponentially growing WT and Δ dnaA cells. Genomic DNA was extracted from cells 24 h after inoculation ( Figure 4B ) and analyzed using an Illumina MiSeq System. The number of reads (divided by the number of total reads) of the growing (replicating) cells was normalized by that of the stationary phase (non-replicating) cells at each genomic position. 1-kb window (left) and 100-kb window (right) of WT and Δ dnaA clones.

Techniques Used: Periodic Counter-current Chromatography, High Throughput Screening Assay, Clone Assay

5) Product Images from "Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing"

Article Title: Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing

Journal: mSphere

doi: 10.1128/mSphere.01202-20

Overview of the analysis strategies used in this study. DNAs from different sample types with increasing complexity (i.e., 3 mock communities and 33 human stool samples) were extracted. Amplicons were generated using different primer pairs targeting different V-regions and sequenced on an Illumina MiSeq. Afterwards, the impacts of different clustering approaches and reference databases on the microbial profiles were investigated.
Figure Legend Snippet: Overview of the analysis strategies used in this study. DNAs from different sample types with increasing complexity (i.e., 3 mock communities and 33 human stool samples) were extracted. Amplicons were generated using different primer pairs targeting different V-regions and sequenced on an Illumina MiSeq. Afterwards, the impacts of different clustering approaches and reference databases on the microbial profiles were investigated.

Techniques Used: Generated

6) Product Images from "Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic"

Article Title: Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic

Journal: Viruses

doi: 10.3390/v14020366

Whole-genome sequencing of SARS-CoV-2 isolates from early in the pandemic in New Zealand. ( A ) Coverage, i.e., number of reads per nucleotide position obtained by deep sequencing the patient-derived samples (blue) and SARS-CoV-2 isolates (red) using the MiSeq platform (Illumina). The position relative to the SARS-CoV-2 isolate Wuhan-Hu-1 NC_045512 is indicated. Maximum Likelihood phylogenetic trees were constructed using ( B ) whole-genome SARS-CoV-2 consensus sequences obtained from all seven patient-derived samples and the seven SARS-CoV-2 isolates, rooted with the Wuhan-Hu-1 NC_045512 sequence, ( C ) whole-genome consensus sequences of the seven SARS-CoV-2 isolates and 28 SARS-like betacoronaviruses, and ( D ) whole-genome consensus sequences of the seven SARS-CoV-2 isolates and 70 contemporary SARS-CoV-2 sequences from different lineages obtained from GISAID database ( https://www.gisaid.org/ , accessed on 6 June 2020). Each color-coded dot represents SARS-CoV-2 GISAID clades. Bootstrap resampling (1000 data sets) of the multiple alignments tested the statistical robustness of the trees, with percentage values above 75% indicated by an asterisk. s/nt, substitutions per nucleotide. ( E ) Classification of the seven SARS-CoV-2 whole-genome sequences using a Nextstrain ( https://nextstrain.org/ncov/ , accessed on 30 April 2020), b PANGO Lineages ( https://cov-lineages.org/index.html , accessed on 30 April 2020), and c GISAID database ( https://www.gisaid.org/ , accessed on 30 April 2020).
Figure Legend Snippet: Whole-genome sequencing of SARS-CoV-2 isolates from early in the pandemic in New Zealand. ( A ) Coverage, i.e., number of reads per nucleotide position obtained by deep sequencing the patient-derived samples (blue) and SARS-CoV-2 isolates (red) using the MiSeq platform (Illumina). The position relative to the SARS-CoV-2 isolate Wuhan-Hu-1 NC_045512 is indicated. Maximum Likelihood phylogenetic trees were constructed using ( B ) whole-genome SARS-CoV-2 consensus sequences obtained from all seven patient-derived samples and the seven SARS-CoV-2 isolates, rooted with the Wuhan-Hu-1 NC_045512 sequence, ( C ) whole-genome consensus sequences of the seven SARS-CoV-2 isolates and 28 SARS-like betacoronaviruses, and ( D ) whole-genome consensus sequences of the seven SARS-CoV-2 isolates and 70 contemporary SARS-CoV-2 sequences from different lineages obtained from GISAID database ( https://www.gisaid.org/ , accessed on 6 June 2020). Each color-coded dot represents SARS-CoV-2 GISAID clades. Bootstrap resampling (1000 data sets) of the multiple alignments tested the statistical robustness of the trees, with percentage values above 75% indicated by an asterisk. s/nt, substitutions per nucleotide. ( E ) Classification of the seven SARS-CoV-2 whole-genome sequences using a Nextstrain ( https://nextstrain.org/ncov/ , accessed on 30 April 2020), b PANGO Lineages ( https://cov-lineages.org/index.html , accessed on 30 April 2020), and c GISAID database ( https://www.gisaid.org/ , accessed on 30 April 2020).

Techniques Used: Sequencing, Derivative Assay, Construct

Microbiota analysis of patient-derived nasopharyngeal samples and SARS-CoV-2 isolates. Sequences obtained with the MiSeq (Illumina) platform were analyzed with CZ ID ( https://czid.org/ accessed on 2 June 2020) to detect and quantify bacteria and viruses. ( A ) Taxon heatmaps represent the number of viral (top) and bacterial (bottom) sequences identified in both sets of samples. Full viral names are included, while bacteria are listed as genera. Heatmap scales in thousands (K) of total non-redundant protein reads per million are included. ( B ) Comparison of the abundance of SARS-CoV-2, other viruses, and bacteria sequences, quantified as total non-redundant protein reads per million, between the patient-derived nasopharyngeal samples (Patient Derived) and SARS-CoV-2 isolates (Virus Isolates). Wilcoxon–Mann–Whitney test was used to compare the abundance between both sets of samples. **** p
Figure Legend Snippet: Microbiota analysis of patient-derived nasopharyngeal samples and SARS-CoV-2 isolates. Sequences obtained with the MiSeq (Illumina) platform were analyzed with CZ ID ( https://czid.org/ accessed on 2 June 2020) to detect and quantify bacteria and viruses. ( A ) Taxon heatmaps represent the number of viral (top) and bacterial (bottom) sequences identified in both sets of samples. Full viral names are included, while bacteria are listed as genera. Heatmap scales in thousands (K) of total non-redundant protein reads per million are included. ( B ) Comparison of the abundance of SARS-CoV-2, other viruses, and bacteria sequences, quantified as total non-redundant protein reads per million, between the patient-derived nasopharyngeal samples (Patient Derived) and SARS-CoV-2 isolates (Virus Isolates). Wilcoxon–Mann–Whitney test was used to compare the abundance between both sets of samples. **** p

Techniques Used: Derivative Assay, MANN-WHITNEY

7) Product Images from "P.F508del editing in cells from cystic fibrosis patients"

Article Title: P.F508del editing in cells from cystic fibrosis patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0242094

CFTR gene editing in iPSCs. Cells were transfected with different combinations of Cas9, sgRNA and ssODN; DNA (n = 2 or n = 1) was isolated after 48–72 hours and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. The Kruskal-Wallis test was used to analyze differences. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM. iPSCs–induced pluripotent stem cells.
Figure Legend Snippet: CFTR gene editing in iPSCs. Cells were transfected with different combinations of Cas9, sgRNA and ssODN; DNA (n = 2 or n = 1) was isolated after 48–72 hours and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. The Kruskal-Wallis test was used to analyze differences. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM. iPSCs–induced pluripotent stem cells.

Techniques Used: Transfection, Isolation, Amplification, Plasmid Preparation, Sequencing, Software, Non-Homologous End Joining

CFTR gene editing in CFTE29o- cells. Cells were transfected with various combinations of Cas9, sgRNA and ssODN. DNA (n = 2) was isolated 48–72 hours after transfection and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM.
Figure Legend Snippet: CFTR gene editing in CFTE29o- cells. Cells were transfected with various combinations of Cas9, sgRNA and ssODN. DNA (n = 2) was isolated 48–72 hours after transfection and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM.

Techniques Used: Transfection, Isolation, Amplification, Plasmid Preparation, Sequencing, Software, Non-Homologous End Joining

8) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.
Figure Legend Snippet: Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.

Techniques Used:

9) Product Images from "Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk"

Article Title: Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

Journal: Microorganisms

doi: 10.3390/microorganisms9061251

Overview of the experimental procedure. Mock communities of known composition and bovine raw milk samples were used for the microbial gDNA extraction (grey above). The samples were prepared for short amplicon 16S rRNA gene sequencing (blue) and full-length sequencing using the 16S/18S kit targeting the SSU rRNA (orange). After the libraries were prepared, cleaned, and attested to be of good quality, the sequencing was performed on an Illumina MiSeq (grey, below). The execution time estimations in hours are shown for all steps.
Figure Legend Snippet: Overview of the experimental procedure. Mock communities of known composition and bovine raw milk samples were used for the microbial gDNA extraction (grey above). The samples were prepared for short amplicon 16S rRNA gene sequencing (blue) and full-length sequencing using the 16S/18S kit targeting the SSU rRNA (orange). After the libraries were prepared, cleaned, and attested to be of good quality, the sequencing was performed on an Illumina MiSeq (grey, below). The execution time estimations in hours are shown for all steps.

Techniques Used: Amplification, Sequencing

10) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.
Figure Legend Snippet: Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.

Techniques Used:

11) Product Images from "Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid"

Article Title: Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

Journal: bioRxiv

doi: 10.1101/176529

Sample collection, DNA isolation, and shotgun metagenomic sequencing. A) I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n=7) and intravitreal injection (n=7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B), and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using II.) Cultivation-based analyses and III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. B) More details to steps II.) and III.): II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA), and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12-14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Figure 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.
Figure Legend Snippet: Sample collection, DNA isolation, and shotgun metagenomic sequencing. A) I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n=7) and intravitreal injection (n=7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B), and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using II.) Cultivation-based analyses and III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. B) More details to steps II.) and III.): II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA), and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12-14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Figure 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.

Techniques Used: DNA Extraction, Sequencing, Injection, Shotgun Sequencing, Mass Spectrometry, Polymerase Chain Reaction

12) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.
Figure Legend Snippet: Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.

Techniques Used:

13) Product Images from "Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors). Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors"

Article Title: Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors). Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors

Journal: Physiological Reports

doi: 10.14814/phy2.15344

Estrogen receptor activation decreases Firmicutes and increases Bacteroidetes . After 10 weeks of high‐fat feeding in Ovariectomized female rats, fecal samples were collected. 16S rDNA sequencing was performed using the MiSeq system.
Figure Legend Snippet: Estrogen receptor activation decreases Firmicutes and increases Bacteroidetes . After 10 weeks of high‐fat feeding in Ovariectomized female rats, fecal samples were collected. 16S rDNA sequencing was performed using the MiSeq system.

Techniques Used: Activation Assay, Sequencing

14) Product Images from "Evaluation of EPISEQ SARS-CoV-2 and a Fully Integrated Application to Identify SARS-CoV-2 Variants from Several Next-Generation Sequencing Approaches"

Article Title: Evaluation of EPISEQ SARS-CoV-2 and a Fully Integrated Application to Identify SARS-CoV-2 Variants from Several Next-Generation Sequencing Approaches

Journal: Viruses

doi: 10.3390/v14081674

Percentage of reference genome coverage determined by EPISEQ SARS-CoV-2 upon whole-genome sequencing with different kits and sequencing platforms ( Table 1 ). ( a ) Pre-omicron SARS-CoV-2-positive samples (including seven 20A-G (EU1), four alpha, two beta, one gamma, six delta and one Eta SARS-CoV-2 variants; n = 21) were sequenced using four different commercial kits and primer pools (ARTIC v3, v4, v4.1 and VSS v1) on two NGS platforms (Illumina MiSeq and ONT GridION), generating 168 sequencing results. ( b ) Omicron-positive SARS-CoV-2 samples (including nine BA.1 and 10 BA.2 omicron sub-variants; n = 19) were sequenced using two different commercial kits and primer pools (ARTIC v4.1 and VSS v2) on the same two NGS platforms (Illumina MiSeq and ONT GridION), generating 76 sequencing results. A total of 244 raw NGS data were generated and analysed using EPISEQ SARS-CoV-2. The dashed line indicates 95% coverage (quality control criteria). Abbreviations: Av4.1, ARTIC kit version 4.1; Av4, ARTIC kit version 4; Av3, ARTIC kit version 3; Illumina, San Diego, USA Illumina sequencing; ONT, Oxford, UK Oxford Nanopore Technologies sequencing; VSSv1, VarSkip Short kit version 1; VSSv2, VarSkip Short kit version 2.
Figure Legend Snippet: Percentage of reference genome coverage determined by EPISEQ SARS-CoV-2 upon whole-genome sequencing with different kits and sequencing platforms ( Table 1 ). ( a ) Pre-omicron SARS-CoV-2-positive samples (including seven 20A-G (EU1), four alpha, two beta, one gamma, six delta and one Eta SARS-CoV-2 variants; n = 21) were sequenced using four different commercial kits and primer pools (ARTIC v3, v4, v4.1 and VSS v1) on two NGS platforms (Illumina MiSeq and ONT GridION), generating 168 sequencing results. ( b ) Omicron-positive SARS-CoV-2 samples (including nine BA.1 and 10 BA.2 omicron sub-variants; n = 19) were sequenced using two different commercial kits and primer pools (ARTIC v4.1 and VSS v2) on the same two NGS platforms (Illumina MiSeq and ONT GridION), generating 76 sequencing results. A total of 244 raw NGS data were generated and analysed using EPISEQ SARS-CoV-2. The dashed line indicates 95% coverage (quality control criteria). Abbreviations: Av4.1, ARTIC kit version 4.1; Av4, ARTIC kit version 4; Av3, ARTIC kit version 3; Illumina, San Diego, USA Illumina sequencing; ONT, Oxford, UK Oxford Nanopore Technologies sequencing; VSSv1, VarSkip Short kit version 1; VSSv2, VarSkip Short kit version 2.

Techniques Used: Sequencing, Next-Generation Sequencing, Generated

15) Product Images from "Comparative analysis of microbial diversity in two hot springs of Bakreshwar, West Bengal, India"

Article Title: Comparative analysis of microbial diversity in two hot springs of Bakreshwar, West Bengal, India

Journal: Genomics Data

doi: 10.1016/j.gdata.2017.04.001

Read Quality Plot in Illumina MiSeq. This plot describes the average quality pattern by showing on the X-axis quality thresholds and on the Y-axis the percentage of reads that exceed that quality level.
Figure Legend Snippet: Read Quality Plot in Illumina MiSeq. This plot describes the average quality pattern by showing on the X-axis quality thresholds and on the Y-axis the percentage of reads that exceed that quality level.

Techniques Used:

16) Product Images from "Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection"

Article Title: Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection

Journal: Analytical Chemistry

doi: 10.1021/acs.analchem.2c00554

(A) Step-by-step process for AptaFlow. (i) incubating reagents with SARS-CoV-2 sample. (ii) dipping of the test strip into the sample. (iii) washing to reduce nonspecific signals. (iv) enhancing signal to increase sensitivity. (B) Limit of detection of LFA system with UV-inactivated virus (UV) at various concentrations (copies/mL) and heat-inactivated (HI) virus (negative control). Bars indicate mean. Brackets indicate 95% CI. Statistical significance was determined by one-way ANOVA with Dunnett correction. * indicates p
Figure Legend Snippet: (A) Step-by-step process for AptaFlow. (i) incubating reagents with SARS-CoV-2 sample. (ii) dipping of the test strip into the sample. (iii) washing to reduce nonspecific signals. (iv) enhancing signal to increase sensitivity. (B) Limit of detection of LFA system with UV-inactivated virus (UV) at various concentrations (copies/mL) and heat-inactivated (HI) virus (negative control). Bars indicate mean. Brackets indicate 95% CI. Statistical significance was determined by one-way ANOVA with Dunnett correction. * indicates p

Techniques Used: Stripping Membranes, Negative Control

17) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.
Figure Legend Snippet: Overall sensitivity of the Ion S5 and Miseq at different aneuploid levels are displayed in the bar chart, and the table lists individual sensitivity for segmental deletion and whole chromosomal duplication ( 2a ). Overall specificity of the Ion S5 and Miseq are shown ( 2b ). Both the sensitivity and specificity are not significantly different between the two systems.

Techniques Used:

18) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

19) Product Images from "Severe preeclampsia is associated with a higher relative abundance of Prevotella bivia in the vaginal microbiota"

Article Title: Severe preeclampsia is associated with a higher relative abundance of Prevotella bivia in the vaginal microbiota

Journal: Scientific Reports

doi: 10.1038/s41598-020-75534-3

Vaginal microbiota community in cases with severe preeclampsia (n = 30) and control women (n = 30). ( a ) Richness and diversity indices of the vaginal microbiota were calculated from 16S ribosomal RNA gene sequencing of the V3–V4 region on a MiSeq platform. ( b ) A principal component analysis plot was constructed using unweighted UniFrac analysis.
Figure Legend Snippet: Vaginal microbiota community in cases with severe preeclampsia (n = 30) and control women (n = 30). ( a ) Richness and diversity indices of the vaginal microbiota were calculated from 16S ribosomal RNA gene sequencing of the V3–V4 region on a MiSeq platform. ( b ) A principal component analysis plot was constructed using unweighted UniFrac analysis.

Techniques Used: Sequencing, Construct

20) Product Images from "High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq"

Article Title: High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq

Journal: Scientific Reports

doi: 10.1038/s41598-021-98318-9

Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.
Figure Legend Snippet: Cell lines are mixed to create multiple levels of aneuploidy. The calculated copy number at the affected aneuploid region displayed correlation with the aneuploid percentage using automatic identification via the Ion Reporter on the Ion S5 system ( 1a ), and using manual identification via the BlueFuse Multi on the Miseq system ( 1b ). As the number of aneuploid cells in the mixtures increases, the copy number of the regions with segmental deletion or whole-chromosome duplication decreases or increases on both the NGS systems.

Techniques Used: Next-Generation Sequencing

21) Product Images from "Dysbiosis-Associated Polyposis of the Colon—Cap Polyposis"

Article Title: Dysbiosis-Associated Polyposis of the Colon—Cap Polyposis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00918

Fecal microbiota analysis in a patient with cap polyposis. (A) Stool samples were obtained from a patient with cap polyposis prior to, 1 week and 6 months post-antibiotic treatment. DNA samples extracted from the stool specimens were subjected to polymerase chain reaction for the amplification of the 16S ribosomal RNA (16S rRNA) V3 and V4 regions. We performed 16S rRNA sequencing using the MiSeq system. The relative abundance of different bacterial taxa at the genus level in each sample has been shown. (B) Comparative analysis of the taxonomic composition of the fecal microbial community at the genus level. Relative abundance of the genera has been shown as a percentage.
Figure Legend Snippet: Fecal microbiota analysis in a patient with cap polyposis. (A) Stool samples were obtained from a patient with cap polyposis prior to, 1 week and 6 months post-antibiotic treatment. DNA samples extracted from the stool specimens were subjected to polymerase chain reaction for the amplification of the 16S ribosomal RNA (16S rRNA) V3 and V4 regions. We performed 16S rRNA sequencing using the MiSeq system. The relative abundance of different bacterial taxa at the genus level in each sample has been shown. (B) Comparative analysis of the taxonomic composition of the fecal microbial community at the genus level. Relative abundance of the genera has been shown as a percentage.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

22) Product Images from "A novel bioluminescent herpes simplex virus 1 for in vivo monitoring of herpes simplex encephalitis"

Article Title: A novel bioluminescent herpes simplex virus 1 for in vivo monitoring of herpes simplex encephalitis

Journal: Scientific Reports

doi: 10.1038/s41598-021-98047-z

CRISPR-Cas9-mediated generation of recombinant HSV-1 (rHSV-1). Schematic presentation of two workflows: ( i ) gRNA validation and ( ii ) rHSV-1 production and the validation. ( i ) gRNA validation: Following the transfection/infection step (P1: CRISPR-Cas9 vector/ HSV-1 strain H25), collected supernatants were diluted and used to infect naïve Vero cells. Supernatant was collected and viral DNA was amplified. PCR products from the amplification of UL26-UL27 intergenic region were first gel-purified, then Sanger sequenced for NHEJ-mediated indel mutation analysis. ( ii ) rHSV-1 production and validation: Following the transfection/infection step (a mixture of plasmids (P1: CRISPR-Cas9 vector and P2: The HDR (homology-directed repair) donor plasmid)/HSV-1 H25 strain), collected supernatants were used to infect naïve Vero cells. tdTomato + rHSV-1 infected cells were isolated and used to infect naïve Vero cells. Five rounds of isolation/infection process were performed to eliminate all WT HSV-1 virions from rHSV-1 viral production. Supernatant was collected and DNA extraction was performed. PCR products from multiplex PCR were gel-purified. Two viral DNAs (WT HSV-1 H25 strain, rHSV-1) and gel-purified Multiplex PCR products were deep-sequenced by using Illumina MiSeq. Bioinformatic analysis was performed for whole-genome construction of both viruses. Images were created using Adobe Illustrator (version 25.3.1/ https://www.adobe.com/ ).
Figure Legend Snippet: CRISPR-Cas9-mediated generation of recombinant HSV-1 (rHSV-1). Schematic presentation of two workflows: ( i ) gRNA validation and ( ii ) rHSV-1 production and the validation. ( i ) gRNA validation: Following the transfection/infection step (P1: CRISPR-Cas9 vector/ HSV-1 strain H25), collected supernatants were diluted and used to infect naïve Vero cells. Supernatant was collected and viral DNA was amplified. PCR products from the amplification of UL26-UL27 intergenic region were first gel-purified, then Sanger sequenced for NHEJ-mediated indel mutation analysis. ( ii ) rHSV-1 production and validation: Following the transfection/infection step (a mixture of plasmids (P1: CRISPR-Cas9 vector and P2: The HDR (homology-directed repair) donor plasmid)/HSV-1 H25 strain), collected supernatants were used to infect naïve Vero cells. tdTomato + rHSV-1 infected cells were isolated and used to infect naïve Vero cells. Five rounds of isolation/infection process were performed to eliminate all WT HSV-1 virions from rHSV-1 viral production. Supernatant was collected and DNA extraction was performed. PCR products from multiplex PCR were gel-purified. Two viral DNAs (WT HSV-1 H25 strain, rHSV-1) and gel-purified Multiplex PCR products were deep-sequenced by using Illumina MiSeq. Bioinformatic analysis was performed for whole-genome construction of both viruses. Images were created using Adobe Illustrator (version 25.3.1/ https://www.adobe.com/ ).

Techniques Used: CRISPR, Recombinant, Transfection, Infection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Purification, Non-Homologous End Joining, Mutagenesis, Isolation, DNA Extraction, Multiplex Assay

23) Product Images from "Comparative analysis of microbial diversity in two hot springs of Bakreshwar, West Bengal, India"

Article Title: Comparative analysis of microbial diversity in two hot springs of Bakreshwar, West Bengal, India

Journal: Genomics Data

doi: 10.1016/j.gdata.2017.04.001

Read Quality Plot in Illumina MiSeq. This plot describes the average quality pattern by showing on the X-axis quality thresholds and on the Y-axis the percentage of reads that exceed that quality level.
Figure Legend Snippet: Read Quality Plot in Illumina MiSeq. This plot describes the average quality pattern by showing on the X-axis quality thresholds and on the Y-axis the percentage of reads that exceed that quality level.

Techniques Used:

24) Product Images from "Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid"

Article Title: Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

Journal: Scientific Reports

doi: 10.1038/s41598-018-22416-4

Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.
Figure Legend Snippet: Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.

Techniques Used: DNA Extraction, Sequencing, Injection, Shotgun Sequencing, Mass Spectrometry, Polymerase Chain Reaction

25) Product Images from "Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol"

Article Title: Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol

Journal: bioRxiv

doi: 10.1101/183780

Two examples of chimeric REPLI-g generated nanopore reads from virus-positive mosquito pool sample 4.1. Illumina MiSeq reads that mapped to any strain in the custom VEEV database were isolated and re-mapped to REPLI-g generated nanopore reads that also aligned to VEEV references in CLC-Bio Genomics Workbench v. 10.0.1. One would expect generally uniform distribution of re-mapped MiSeq reads across VEEV-associated nanopore reads. However, MiSeq reads are observed to align with specific regions of nanopore reads, and are absent from other regions, indicating chimerism in REPLI-g generated nanopore reads. (A) The purple-highlighted region of Channel_284_read_6073 was BLASTed against the nt database and the highest associated hits were for the mosquito Culex qainqaefasciatas and Drosophila spp. (B) The purple highlighted region of Channel_102_read_4542 returned C.ulex quinquefasciatus and Aedes aegypti as top hits in its BLAST result.
Figure Legend Snippet: Two examples of chimeric REPLI-g generated nanopore reads from virus-positive mosquito pool sample 4.1. Illumina MiSeq reads that mapped to any strain in the custom VEEV database were isolated and re-mapped to REPLI-g generated nanopore reads that also aligned to VEEV references in CLC-Bio Genomics Workbench v. 10.0.1. One would expect generally uniform distribution of re-mapped MiSeq reads across VEEV-associated nanopore reads. However, MiSeq reads are observed to align with specific regions of nanopore reads, and are absent from other regions, indicating chimerism in REPLI-g generated nanopore reads. (A) The purple-highlighted region of Channel_284_read_6073 was BLASTed against the nt database and the highest associated hits were for the mosquito Culex qainqaefasciatas and Drosophila spp. (B) The purple highlighted region of Channel_102_read_4542 returned C.ulex quinquefasciatus and Aedes aegypti as top hits in its BLAST result.

Techniques Used: Generated, Isolation

26) Product Images from "The Mycobiota of High Altitude Pear Orchards Soil in Colombia"

Article Title: The Mycobiota of High Altitude Pear Orchards Soil in Colombia

Journal: Biology

doi: 10.3390/biology10101002

Principal coordinate analysis (PCoA) based on Bray–Curtis distances matrix of Illumina MiSeq sequencing fungal data of soil samples taken from pear orchards in Soracá (SR) and Nuevo Colón (steep slope plot at higher altitude: NC-A; flat plot at lower altitude: NC-B).
Figure Legend Snippet: Principal coordinate analysis (PCoA) based on Bray–Curtis distances matrix of Illumina MiSeq sequencing fungal data of soil samples taken from pear orchards in Soracá (SR) and Nuevo Colón (steep slope plot at higher altitude: NC-A; flat plot at lower altitude: NC-B).

Techniques Used: Sequencing

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    Illumina Inc miseq
    Mutation analysis. The mutation CDS 456 A > T in KRAS was detected with <t>MiSeq</t> using a <t>TruSight</t> One Panel and was resequenced by Sanger sequencing.
    Miseq, 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
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    illumina inc bulk pcr miseq sequencing
    CAG frequency distributions obtained by <t>MiSeq</t> or PacBio SMRT sequencing of <t>bulk-PCR</t> products obtained for different tissues of one 6-week-old and one 117-week-old R6/2 mouse with ∼110 CAGs. MiSeq sequencing data in white and PacBio SMRT sequencing data in grey. The dotted line on the MiSeq sequencing data panels indicates 123 CAGs, which is the theoretical maximum number of CAGs that could have been sequenced using the PCR primer pair (31329/33934) and a 400 nt MiSeq read.
    Bulk Pcr Miseq Sequencing, 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
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    Illumina Inc miseq reagent kit
    Immunoglobulin sequencing from turquoise-killiñsh total-RNA samples. Each sample undergoes reverse-transcription with template switching to attach a 5’ adaptor sequence and unique molecular identifier (UMI), followed by multiple rounds of PCR amplification and addition of <t>Illumina</t> sequencing adaptors. Libraries are then pooled, undergo size selection, and are sequenced on an Illumina <t>MiSeq</t> sequencing machine
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    Illumina Inc illumina miseq sequencing
    The co-occurrence networks of bacteria and fungi based on 16S rRNA and ITS <t>MiSeq</t> <t>Illumina</t> sequences. Connections materialize strong (Spearman’s ǀρǀ > 0.85) and significant ( P
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    Mutation analysis. The mutation CDS 456 A > T in KRAS was detected with MiSeq using a TruSight One Panel and was resequenced by Sanger sequencing.

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: Induction of Noonan syndrome-specific human-induced pluripotent stem cells under serum-, feeder-, and integration-free conditions

    doi: 10.1007/s11626-020-00515-9

    Figure Lengend Snippet: Mutation analysis. The mutation CDS 456 A > T in KRAS was detected with MiSeq using a TruSight One Panel and was resequenced by Sanger sequencing.

    Article Snippet: To reveal mutations, targeted resequencing was performed with MiSeq (Illumina, San Diego, CA) using a TruSight One Panel, which is designed to comprehensively cover more than 4800 genes involved in diseases according to the manufacturer’s protocol.

    Techniques: Mutagenesis, Sequencing

    CAG frequency distributions obtained by MiSeq or PacBio SMRT sequencing of bulk-PCR products obtained for different tissues of one 6-week-old and one 117-week-old R6/2 mouse with ∼110 CAGs. MiSeq sequencing data in white and PacBio SMRT sequencing data in grey. The dotted line on the MiSeq sequencing data panels indicates 123 CAGs, which is the theoretical maximum number of CAGs that could have been sequenced using the PCR primer pair (31329/33934) and a 400 nt MiSeq read.

    Journal: Journal of Huntington's Disease

    Article Title: Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

    doi: 10.3233/JHD-200433

    Figure Lengend Snippet: CAG frequency distributions obtained by MiSeq or PacBio SMRT sequencing of bulk-PCR products obtained for different tissues of one 6-week-old and one 117-week-old R6/2 mouse with ∼110 CAGs. MiSeq sequencing data in white and PacBio SMRT sequencing data in grey. The dotted line on the MiSeq sequencing data panels indicates 123 CAGs, which is the theoretical maximum number of CAGs that could have been sequenced using the PCR primer pair (31329/33934) and a 400 nt MiSeq read.

    Article Snippet: Read depth must be considered when directly comparing the results obtained by bulk-PCR PacBio SMRT sequencing and bulk-PCR MiSeq sequencing.

    Techniques: Sequencing, Polymerase Chain Reaction

    SP-PCR can detect very large HTT CAG somatic expansions (≥90 CAGs) that cannot be detected using bulk-PCR approaches. A) Representative small pool PCR autoradiograph from 150 pg template DNA obtained for the striatum of the 117-week-old R6/2 mouse with ∼55 CAGs. The number of CAG repeats, equivalent to each molecular weight marker (left) and the boundaries of the categories represented in panel A (right), is indicated. The boundaries of the categories represented in panel A (right) are also indicated by white dashed lines. B) Percentage of large (≥70 CAGs) HTT CAG somatic expansions detected by SP-PCR (black to white gradient), or bulk-PCR capillary electrophoresis (black), bulk-PCR MiSeq (white), bulk-PCR PacBio SMRT (grey) in the striatum of the 117-week-old R6/2 mouse with progenitor allele ∼55 CAGs. C: HTT CAG somatic expansions > 90 CAGs from panel B. Error bars indicate the 95% confidence intervals (they could not be estimated for the bulk-PCR capillary electrophoresis because the fluorescence units measured cannot be transformed into a count of PCR products detected).

    Journal: Journal of Huntington's Disease

    Article Title: Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

    doi: 10.3233/JHD-200433

    Figure Lengend Snippet: SP-PCR can detect very large HTT CAG somatic expansions (≥90 CAGs) that cannot be detected using bulk-PCR approaches. A) Representative small pool PCR autoradiograph from 150 pg template DNA obtained for the striatum of the 117-week-old R6/2 mouse with ∼55 CAGs. The number of CAG repeats, equivalent to each molecular weight marker (left) and the boundaries of the categories represented in panel A (right), is indicated. The boundaries of the categories represented in panel A (right) are also indicated by white dashed lines. B) Percentage of large (≥70 CAGs) HTT CAG somatic expansions detected by SP-PCR (black to white gradient), or bulk-PCR capillary electrophoresis (black), bulk-PCR MiSeq (white), bulk-PCR PacBio SMRT (grey) in the striatum of the 117-week-old R6/2 mouse with progenitor allele ∼55 CAGs. C: HTT CAG somatic expansions > 90 CAGs from panel B. Error bars indicate the 95% confidence intervals (they could not be estimated for the bulk-PCR capillary electrophoresis because the fluorescence units measured cannot be transformed into a count of PCR products detected).

    Article Snippet: Read depth must be considered when directly comparing the results obtained by bulk-PCR PacBio SMRT sequencing and bulk-PCR MiSeq sequencing.

    Techniques: Polymerase Chain Reaction, Autoradiography, Molecular Weight, Marker, Electrophoresis, Fluorescence, Transformation Assay

    Qualitative assessment of somatic mosaicism comparing CAG frequency distributions obtained by capillary electrophoresis, MiSeq or PacBio SMRT sequencing of bulk-PCR products obtained for different tissues of one 6-week-old and one 117-week-old R6/2 mouse with ∼55 CAGs. Capillary electrophoresis data in black, MiSeq sequencing data in white and PacBio SMRT sequencing data in grey.

    Journal: Journal of Huntington's Disease

    Article Title: Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

    doi: 10.3233/JHD-200433

    Figure Lengend Snippet: Qualitative assessment of somatic mosaicism comparing CAG frequency distributions obtained by capillary electrophoresis, MiSeq or PacBio SMRT sequencing of bulk-PCR products obtained for different tissues of one 6-week-old and one 117-week-old R6/2 mouse with ∼55 CAGs. Capillary electrophoresis data in black, MiSeq sequencing data in white and PacBio SMRT sequencing data in grey.

    Article Snippet: Read depth must be considered when directly comparing the results obtained by bulk-PCR PacBio SMRT sequencing and bulk-PCR MiSeq sequencing.

    Techniques: Electrophoresis, Sequencing, Polymerase Chain Reaction

    Representative sequence alignments of the 400 nt MiSeq reads (A and B), PacBio CCS reads (C and D) and PacBio subreads (E and F) uniquely aligned (i.e., reads not discarded post alignment) to a synthetic reference sequence with 115 CAGs. Alignments shown correspond to 30 sequencing reads obtained from the tail at weaning of the 20-week-old mouse with ∼110 CAGs. The part of the alignment shown corresponds to the four nucleotides in the immediate 5’–flank of the HTT CAG repeat, followed by the first 20 CAGs (A, C and E), as well as the last 7 CAGs followed by (CAACAG) 1 (CCGCCA) 1 (CCG) 7 (CCT) 2 and the four nucleotides in the immediate 3’-flank of that sequence (B, D and F). Note that the last nucleotide sequenced for the sample with the 400 nt MiSeq reads end was the first C of the seventh CCG (B). The white box on the right-hand side of panel B represents the part of the PCR products containing 115 CAGs that could not be sequenced using 400 nt MiSeq reads.

    Journal: Journal of Huntington's Disease

    Article Title: Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

    doi: 10.3233/JHD-200433

    Figure Lengend Snippet: Representative sequence alignments of the 400 nt MiSeq reads (A and B), PacBio CCS reads (C and D) and PacBio subreads (E and F) uniquely aligned (i.e., reads not discarded post alignment) to a synthetic reference sequence with 115 CAGs. Alignments shown correspond to 30 sequencing reads obtained from the tail at weaning of the 20-week-old mouse with ∼110 CAGs. The part of the alignment shown corresponds to the four nucleotides in the immediate 5’–flank of the HTT CAG repeat, followed by the first 20 CAGs (A, C and E), as well as the last 7 CAGs followed by (CAACAG) 1 (CCGCCA) 1 (CCG) 7 (CCT) 2 and the four nucleotides in the immediate 3’-flank of that sequence (B, D and F). Note that the last nucleotide sequenced for the sample with the 400 nt MiSeq reads end was the first C of the seventh CCG (B). The white box on the right-hand side of panel B represents the part of the PCR products containing 115 CAGs that could not be sequenced using 400 nt MiSeq reads.

    Article Snippet: Read depth must be considered when directly comparing the results obtained by bulk-PCR PacBio SMRT sequencing and bulk-PCR MiSeq sequencing.

    Techniques: Sequencing, Polymerase Chain Reaction

    Method summary for somatic mosaicism quantification at the level of a single molecule in HD. A) Generalised schematics for CRISPR/Casp9-mediated targeted enrichment of HTT locus for single-molecule long-read sequencing (i.e., no-amp targeted sequencing). Following DNA fragmentation and DNA molecule protection by adapter ligation or de-phosphorylation, CRISPR/Cas9 and locus-specific guide RNAs are used to selectively cut across the region of interest. While undigested DNA fragment ends are still protected, sequencing adapters are ligated to the Cas9 digestion product. Sequencing is then done on the appropriate single-molecule long-read sequencing platform such as PacBio SMRT or Oxford Nanopore Technologies (ONT). No-amp targeted sequencing studies of repeat expansions have used one or two Cas9 cuts with PacBio sequencing [ 31, 49, 57, 59 ] or ONT sequencing [ 58 ] respectively. Single-molecule sequencing read output can then be used to build the somatic mosaicism profile. B) The general method for amplicon sequencing of barcoded single molecules. Several methods for single-molecule barcoding exist, including one-cycle PCR using hairpin-protected primers with degenerate tags or region capture by barcoded molecular inversion probes. Following barcoding, sequencing adapters are incorporated into the uniquely tagged molecules through PCR with overhang primers. The resulting amplicon library is then sequenced on the platform of interest, including Illumina MiSeq or PacBio, depending on the amplicon length and the desired throughput. Resulting reads are grouped by barcode family, and the repeat length of the original molecule for each family is determined to build the real somatic mosaicism profile per sample.

    Journal: Journal of Huntington's Disease

    Article Title: Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

    doi: 10.3233/JHD-200433

    Figure Lengend Snippet: Method summary for somatic mosaicism quantification at the level of a single molecule in HD. A) Generalised schematics for CRISPR/Casp9-mediated targeted enrichment of HTT locus for single-molecule long-read sequencing (i.e., no-amp targeted sequencing). Following DNA fragmentation and DNA molecule protection by adapter ligation or de-phosphorylation, CRISPR/Cas9 and locus-specific guide RNAs are used to selectively cut across the region of interest. While undigested DNA fragment ends are still protected, sequencing adapters are ligated to the Cas9 digestion product. Sequencing is then done on the appropriate single-molecule long-read sequencing platform such as PacBio SMRT or Oxford Nanopore Technologies (ONT). No-amp targeted sequencing studies of repeat expansions have used one or two Cas9 cuts with PacBio sequencing [ 31, 49, 57, 59 ] or ONT sequencing [ 58 ] respectively. Single-molecule sequencing read output can then be used to build the somatic mosaicism profile. B) The general method for amplicon sequencing of barcoded single molecules. Several methods for single-molecule barcoding exist, including one-cycle PCR using hairpin-protected primers with degenerate tags or region capture by barcoded molecular inversion probes. Following barcoding, sequencing adapters are incorporated into the uniquely tagged molecules through PCR with overhang primers. The resulting amplicon library is then sequenced on the platform of interest, including Illumina MiSeq or PacBio, depending on the amplicon length and the desired throughput. Resulting reads are grouped by barcode family, and the repeat length of the original molecule for each family is determined to build the real somatic mosaicism profile per sample.

    Article Snippet: Read depth must be considered when directly comparing the results obtained by bulk-PCR PacBio SMRT sequencing and bulk-PCR MiSeq sequencing.

    Techniques: CRISPR, Sequencing, Ligation, De-Phosphorylation Assay, Amplification, Polymerase Chain Reaction

    Immunoglobulin sequencing from turquoise-killiñsh total-RNA samples. Each sample undergoes reverse-transcription with template switching to attach a 5’ adaptor sequence and unique molecular identifier (UMI), followed by multiple rounds of PCR amplification and addition of Illumina sequencing adaptors. Libraries are then pooled, undergo size selection, and are sequenced on an Illumina MiSeq sequencing machine

    Journal: bioRxiv

    Article Title: Antibody repertoire sequencing reveals systemic and mucosal immunosenescence in the short-lived turquoise killifish

    doi: 10.1101/2020.08.21.261248

    Figure Lengend Snippet: Immunoglobulin sequencing from turquoise-killiñsh total-RNA samples. Each sample undergoes reverse-transcription with template switching to attach a 5’ adaptor sequence and unique molecular identifier (UMI), followed by multiple rounds of PCR amplification and addition of Illumina sequencing adaptors. Libraries are then pooled, undergo size selection, and are sequenced on an Illumina MiSeq sequencing machine

    Article Snippet: Finally, following a final round of quality control, the pooled and size-selected libraries were sequenced on an Illumina MiSeq System (MiSeq Reagent Kit v3, 2×300 bp reads, 30% PhiX spike-in), either at the Cologne Center for Genomics (whole-body libraries) or with Admera Health (intestinal libraries).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification, Selection

    The co-occurrence networks of bacteria and fungi based on 16S rRNA and ITS MiSeq Illumina sequences. Connections materialize strong (Spearman’s ǀρǀ > 0.85) and significant ( P

    Journal: Microbiome

    Article Title: Rock substrate rather than black stain alterations drives microbial community structure in the passage of Lascaux Cave

    doi: 10.1186/s40168-018-0599-9

    Figure Lengend Snippet: The co-occurrence networks of bacteria and fungi based on 16S rRNA and ITS MiSeq Illumina sequences. Connections materialize strong (Spearman’s ǀρǀ > 0.85) and significant ( P

    Article Snippet: Libraries were built using 1 μg DNA and Illumina MiSeq sequencing of the PCR products were conducted on 2 ng DNA with specific tag sequences to concurrently sequence different samples on the same run.

    Techniques: