Structured Review

Illumina Inc illumina miseq system
Comparison of sequence analysis databases for assessment of AMR gene amplicon sequencing generated using <t>Illumina</t> <t>MiSeq</t> and Ion torrent. The figures on the top row represts the data from Illumina platform while data of down row represents the Ion Torrent platform. Databases included were (CARD-CLC-CARD database present in CLC genomic workbench microbial genomic module, AR – Antibiotic resistance database, QMI-DB-QIAGEN microbial Insight – AR, CARD-IN – CARD database downloaded from CARD site and run locally)
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1) Product Images from "Comparative analysis of two NGS platforms and different databases for analysis of AMR genes"

Article Title: Comparative analysis of two NGS platforms and different databases for analysis of AMR genes

Journal: bioRxiv

doi: 10.1101/2021.12.27.474239

Comparison of sequence analysis databases for assessment of AMR gene amplicon sequencing generated using Illumina MiSeq and Ion torrent. The figures on the top row represts the data from Illumina platform while data of down row represents the Ion Torrent platform. Databases included were (CARD-CLC-CARD database present in CLC genomic workbench microbial genomic module, AR – Antibiotic resistance database, QMI-DB-QIAGEN microbial Insight – AR, CARD-IN – CARD database downloaded from CARD site and run locally)
Figure Legend Snippet: Comparison of sequence analysis databases for assessment of AMR gene amplicon sequencing generated using Illumina MiSeq and Ion torrent. The figures on the top row represts the data from Illumina platform while data of down row represents the Ion Torrent platform. Databases included were (CARD-CLC-CARD database present in CLC genomic workbench microbial genomic module, AR – Antibiotic resistance database, QMI-DB-QIAGEN microbial Insight – AR, CARD-IN – CARD database downloaded from CARD site and run locally)

Techniques Used: Sequencing, Amplification, Generated

The relative abundance of organisms hosting AMR genes in chicken caecal microbial populations predicted using the CARD database. Organisms represented by ≥1% of the sequence reads generated using Illumina MiSeq or Thermo Fischer Scientific are shown.
Figure Legend Snippet: The relative abundance of organisms hosting AMR genes in chicken caecal microbial populations predicted using the CARD database. Organisms represented by ≥1% of the sequence reads generated using Illumina MiSeq or Thermo Fischer Scientific are shown.

Techniques Used: Sequencing, Generated

The relative sequencing read abundance of AMR genes amplified from chicken caecal microbial populations with ≥1% abundance within equivalent Illumina MiSeq and Thermo Fischer Scientific
Figure Legend Snippet: The relative sequencing read abundance of AMR genes amplified from chicken caecal microbial populations with ≥1% abundance within equivalent Illumina MiSeq and Thermo Fischer Scientific

Techniques Used: Sequencing, Amplification

Heatmap demonstrating occurrence of the top 25 most abundant organisms predicted in chicken caecal microbial populations using the CARD database. Data generated from equivalent Illumina MiSeq (IL) and Ion Torrent (T) AMR amplicon sequencing datasets and plotted using STEMP. Organism abundance illustrated by a color gradient as indicated by the color scale.
Figure Legend Snippet: Heatmap demonstrating occurrence of the top 25 most abundant organisms predicted in chicken caecal microbial populations using the CARD database. Data generated from equivalent Illumina MiSeq (IL) and Ion Torrent (T) AMR amplicon sequencing datasets and plotted using STEMP. Organism abundance illustrated by a color gradient as indicated by the color scale.

Techniques Used: Generated, Amplification, Sequencing

Heatmap demonstrating the abundance of the top 25 AMR genes in 12 chicken caecal samples detected using Illumina MiSeq (IL) or Ion Torrent (T), plotted using STEMP. AMR gene abundance illustrated by a color gradient as indicated by the color scale.
Figure Legend Snippet: Heatmap demonstrating the abundance of the top 25 AMR genes in 12 chicken caecal samples detected using Illumina MiSeq (IL) or Ion Torrent (T), plotted using STEMP. AMR gene abundance illustrated by a color gradient as indicated by the color scale.

Techniques Used:

PERMANOVA analysis of AMR gene and organism occurrence predicted based upon Illumina MiSeq or Ion Torrent sequencing of AMR amplicons from chicken caecal contents. (A) AMR gene PERMANOVA with F-value 1.3421, R 2 value 0.057498 and *p-value
Figure Legend Snippet: PERMANOVA analysis of AMR gene and organism occurrence predicted based upon Illumina MiSeq or Ion Torrent sequencing of AMR amplicons from chicken caecal contents. (A) AMR gene PERMANOVA with F-value 1.3421, R 2 value 0.057498 and *p-value

Techniques Used: Sequencing

2) Product Images from "In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening"

Article Title: In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01524-21

Relative abundances (%) of bacteria identified by MiSeq Illumina from the biofilms on the wooden shelves used to ripen PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheeses in 18 dairy facilities. The amplification occurred on a 464-nucleotide sequence of the V3-V4 region of the 16S rRNA gene. Only taxa occurring at > 0.1% abundance in at least one sample were included. Abbreviations: WSA to -T, wooden shelves from factories A to T, respectively.
Figure Legend Snippet: Relative abundances (%) of bacteria identified by MiSeq Illumina from the biofilms on the wooden shelves used to ripen PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheeses in 18 dairy facilities. The amplification occurred on a 464-nucleotide sequence of the V3-V4 region of the 16S rRNA gene. Only taxa occurring at > 0.1% abundance in at least one sample were included. Abbreviations: WSA to -T, wooden shelves from factories A to T, respectively.

Techniques Used: Amplification, Sequencing

3) Product Images from "In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix"

Article Title: In Vivo Emergence of a Novel Protease Inhibitor Resistance Signature in HIV-1 Matrix

Journal: mBio

doi: 10.1128/mBio.02036-20

Whole-genome HIV haplotype reconstruction using target-enriched NGS Illumina MiSeq data from each time point (baseline, VF1, and VF2), with maximum-likelihood analysis and bootstrap support indicated using 1,000 replicates. Labels on the right are the amino acids at Gag positions 122 and 123.
Figure Legend Snippet: Whole-genome HIV haplotype reconstruction using target-enriched NGS Illumina MiSeq data from each time point (baseline, VF1, and VF2), with maximum-likelihood analysis and bootstrap support indicated using 1,000 replicates. Labels on the right are the amino acids at Gag positions 122 and 123.

Techniques Used: Next-Generation Sequencing

4) Product Images from "Six-Week Endurance Exercise Alters Gut Metagenome That Is not Reflected in Systemic Metabolism in Over-weight Women"

Article Title: Six-Week Endurance Exercise Alters Gut Metagenome That Is not Reflected in Systemic Metabolism in Over-weight Women

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02323

The 16S rRNA gene amplicon sequencing failed to show differences in the microbiota composition between the samples collected before the control period (Pre), after the control period/before exercise (Mid) and after the exercise (Post) period. (A) The genus level abundance analyzed with Illumina MiSeq 16S rRNA gene amplicon sequencing show great inter-individual variation that may have caused the lack of significant differences between the samples collected before the control period (Pre, n = 17), after the control period/before exercise (Mid, n = 17) and after the exercise period (Post, n = 17). (B) PCoA plot of the 16S rRNA gene amplicon sequenced samples before the control period ( n = 17), after the control/before the exercise period ( n = 17) and after the exercise period ( n = 17) shows an important inter-individual variation in the exercise responsiveness.
Figure Legend Snippet: The 16S rRNA gene amplicon sequencing failed to show differences in the microbiota composition between the samples collected before the control period (Pre), after the control period/before exercise (Mid) and after the exercise (Post) period. (A) The genus level abundance analyzed with Illumina MiSeq 16S rRNA gene amplicon sequencing show great inter-individual variation that may have caused the lack of significant differences between the samples collected before the control period (Pre, n = 17), after the control period/before exercise (Mid, n = 17) and after the exercise period (Post, n = 17). (B) PCoA plot of the 16S rRNA gene amplicon sequenced samples before the control period ( n = 17), after the control/before the exercise period ( n = 17) and after the exercise period ( n = 17) shows an important inter-individual variation in the exercise responsiveness.

Techniques Used: Amplification, Sequencing

5) Product Images from "RADA is a main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest"

Article Title: RADA is a main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest

Journal: bioRxiv

doi: 10.1101/856716

No significant effect of the radA mutation on the stability of the plastidial genome. The DNA from a leaf of four independent 4-week-old radA-1 plants and of two WT plants was extracted and sequenced on a MySeq Illumina system (2×150 bp paired-end). The reads corresponding to the cpDNA were extracted and investigated for possible changes in cpDNA stability. ( A ) Normalized cpDNA coverage, showing comparable copy number of the cpDNA in WT and in radA , and no apparent changes in sequences stoichiometry. A schematic representation of the cpDNA is shown above, indicating the large and small single-copy regions (LSC and SSC, respectively) and a single copy of the inverted repeated region (IR). ( B ) Abundance of reads identified as corresponding to rearranged cpDNA molecules (see supplemental figure 7 for a scheme of the analysis pipeline). A slight increase, up to two-fold as compared to WT, was identified in the radA plants.
Figure Legend Snippet: No significant effect of the radA mutation on the stability of the plastidial genome. The DNA from a leaf of four independent 4-week-old radA-1 plants and of two WT plants was extracted and sequenced on a MySeq Illumina system (2×150 bp paired-end). The reads corresponding to the cpDNA were extracted and investigated for possible changes in cpDNA stability. ( A ) Normalized cpDNA coverage, showing comparable copy number of the cpDNA in WT and in radA , and no apparent changes in sequences stoichiometry. A schematic representation of the cpDNA is shown above, indicating the large and small single-copy regions (LSC and SSC, respectively) and a single copy of the inverted repeated region (IR). ( B ) Abundance of reads identified as corresponding to rearranged cpDNA molecules (see supplemental figure 7 for a scheme of the analysis pipeline). A slight increase, up to two-fold as compared to WT, was identified in the radA plants.

Techniques Used: Mutagenesis

6) Product Images from "Rapid High-Throughput Whole-Genome Sequencing of SARS-CoV-2 by Using One-Step Reverse Transcription-PCR Amplification with an Integrated Microfluidic System and Next-Generation Sequencing"

Article Title: Rapid High-Throughput Whole-Genome Sequencing of SARS-CoV-2 by Using One-Step Reverse Transcription-PCR Amplification with an Integrated Microfluidic System and Next-Generation Sequencing

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.02784-20

Whole-genome RT-PCR amplification and Illumina sequencing.
Figure Legend Snippet: Whole-genome RT-PCR amplification and Illumina sequencing.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing

7) Product Images from "Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM)"

Article Title: Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM)

Journal: Journal of Animal Science and Biotechnology

doi: 10.1186/s40104-021-00584-0

Relative abundance of the 15 main bacterial families in the bioreactor medium ( a ) and on the mucin beads ( b ) of the MPigut-IVM during the fermentations #6, 7, 8 and 9 which were subjected to a feed deprivation stress of 48 h, as measured by 16S Illumina sequencing
Figure Legend Snippet: Relative abundance of the 15 main bacterial families in the bioreactor medium ( a ) and on the mucin beads ( b ) of the MPigut-IVM during the fermentations #6, 7, 8 and 9 which were subjected to a feed deprivation stress of 48 h, as measured by 16S Illumina sequencing

Techniques Used: Sequencing

Effects of a feed deprivation stress of 12, 24, and 48 h on the MPigut-IVM microbiota composition. On this figure, the “12h feed deprivation stress”, “24h feed deprivation stress” and “48h feed deprivation stress” correspond to the fermentations #4, 5 and 6, respectively. a Evolution of the redox potential. An example of redox measurement for a control assay is provided one the figure. These data are from the fermentation 2. b Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families in the bioreactor medium of the MPigut-IVM as measured by 16S Illumina sequencing. c Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families on the mucin beads of the MPigut-IVM as measured by 16S Illumina sequencing
Figure Legend Snippet: Effects of a feed deprivation stress of 12, 24, and 48 h on the MPigut-IVM microbiota composition. On this figure, the “12h feed deprivation stress”, “24h feed deprivation stress” and “48h feed deprivation stress” correspond to the fermentations #4, 5 and 6, respectively. a Evolution of the redox potential. An example of redox measurement for a control assay is provided one the figure. These data are from the fermentation 2. b Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families in the bioreactor medium of the MPigut-IVM as measured by 16S Illumina sequencing. c Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families on the mucin beads of the MPigut-IVM as measured by 16S Illumina sequencing

Techniques Used: Control Assay, Sequencing

Set-up and validation of the MPigut-IVM: microbiota composition. a Evolution of the structure and colonization of a mucin bead before (I) and after (II) 48 h of incubation in the MPigut-IVM during the fermentation #1 and observation of the specific adherent microbiota at high magnitude (III IV) by scanning electron microscopy. b Relative abundance of the 15 principal bacterial families in the bioreactor medium of the MPigut-IVM during control assays measured by 16S Illumina sequencing. c Relative abundance of the 15 principal bacterial families on the mucin beads of the MPigut-IVM during control assays measured by 16S Illumina sequencing
Figure Legend Snippet: Set-up and validation of the MPigut-IVM: microbiota composition. a Evolution of the structure and colonization of a mucin bead before (I) and after (II) 48 h of incubation in the MPigut-IVM during the fermentation #1 and observation of the specific adherent microbiota at high magnitude (III IV) by scanning electron microscopy. b Relative abundance of the 15 principal bacterial families in the bioreactor medium of the MPigut-IVM during control assays measured by 16S Illumina sequencing. c Relative abundance of the 15 principal bacterial families on the mucin beads of the MPigut-IVM during control assays measured by 16S Illumina sequencing

Techniques Used: Incubation, Electron Microscopy, Sequencing

8) Product Images from "The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation"

Article Title: The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw045

Nug1 and Dbp10 bind at proximal sites on the intersubunit face of the 60S subunit. CRAC analysis of ( A ) Nug1, ( B ) Nug1 D336N mutant and ( C ) Dbp10. ( A–C ) Illumina-Miseq sequencing results were aligned to the yeast 35S rDNA and plotted. The histogram shows the position and distribution of crosslink sites of Nug1 wild-type (red), Nug1 D336N mutant (green) and Dbp10 (blue) on the 35S rRNA. Position of mature rRNA sequences and spacers are indicated below the x-axis. The y-axis displays number of times each nucleotide was mapped (hits) per 1000 rDNA reads. The location of the peaks in the secondary structure of the rRNA is indicated with helix (H) numbers. The asterisks indicate frequently observed contaminants. Each experiment was performed at least twice, and representative results are shown. ( D ) Overlapping interaction region of Nug1 and Dbp10 on helix 89. Binding sites of Nug1 (red) and Dbp10 (blue) are shown on the secondary structure map of rRNA helices 89–92 ( http://www.rna.ccbb.utexas.edu/ ). ( E ) Mapping of the Nug1 (red) and Dbp10 (blue) crosslink sites on the cryo-EM structure of the early Arx1 pre-60S particle.
Figure Legend Snippet: Nug1 and Dbp10 bind at proximal sites on the intersubunit face of the 60S subunit. CRAC analysis of ( A ) Nug1, ( B ) Nug1 D336N mutant and ( C ) Dbp10. ( A–C ) Illumina-Miseq sequencing results were aligned to the yeast 35S rDNA and plotted. The histogram shows the position and distribution of crosslink sites of Nug1 wild-type (red), Nug1 D336N mutant (green) and Dbp10 (blue) on the 35S rRNA. Position of mature rRNA sequences and spacers are indicated below the x-axis. The y-axis displays number of times each nucleotide was mapped (hits) per 1000 rDNA reads. The location of the peaks in the secondary structure of the rRNA is indicated with helix (H) numbers. The asterisks indicate frequently observed contaminants. Each experiment was performed at least twice, and representative results are shown. ( D ) Overlapping interaction region of Nug1 and Dbp10 on helix 89. Binding sites of Nug1 (red) and Dbp10 (blue) are shown on the secondary structure map of rRNA helices 89–92 ( http://www.rna.ccbb.utexas.edu/ ). ( E ) Mapping of the Nug1 (red) and Dbp10 (blue) crosslink sites on the cryo-EM structure of the early Arx1 pre-60S particle.

Techniques Used: Mutagenesis, Sequencing, Binding Assay

9) Product Images from "Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses"

Article Title: Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Journal: bioRxiv

doi: 10.1101/705632

Overview of library preparation and sequencing kits utilized for preparing viral specimens for sequencing on the Illumina, Ion Torrent PGM and Ion Torrent S5 platforms. Abbreviations for each dataset based on the type of library kit and sequencing kit/cartridge used: NexteraXT 500v2 (MK5), NexteraXT Nano 500v2 (MNN), KAPA HyperPlus 500v2 (MK5), KAPA HyperPlus Nano 500v2 (MKN), KAPA DNA Ion Torrent 316v2 (PD6), KAPA DNA Ion Torrent 318v2 (PD8), KAPA DNA Ion Torrent S5 510 SPRI Size Selection (SDS), KAPA DNA Ion Torrent. 510 E-Gel Size Selection (SDG). *Ion Chef loading is only performed for Ion Torrent sequencing runs.
Figure Legend Snippet: Overview of library preparation and sequencing kits utilized for preparing viral specimens for sequencing on the Illumina, Ion Torrent PGM and Ion Torrent S5 platforms. Abbreviations for each dataset based on the type of library kit and sequencing kit/cartridge used: NexteraXT 500v2 (MK5), NexteraXT Nano 500v2 (MNN), KAPA HyperPlus 500v2 (MK5), KAPA HyperPlus Nano 500v2 (MKN), KAPA DNA Ion Torrent 316v2 (PD6), KAPA DNA Ion Torrent 318v2 (PD8), KAPA DNA Ion Torrent S5 510 SPRI Size Selection (SDS), KAPA DNA Ion Torrent. 510 E-Gel Size Selection (SDG). *Ion Chef loading is only performed for Ion Torrent sequencing runs.

Techniques Used: Sequencing, Selection

10) Product Images from "miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers"

Article Title: miRNome profiling in Duchenne muscular dystrophy; identification of asymptomatic and manifesting female carriers

Journal: Bioscience Reports

doi: 10.1042/BSR20211325

Bioinformatics workflow The workflow shows the bioinformatics pipeline used to analyze the miRNA data generated from Illumina MiSeqDx. The pipeline consists of six components: (1) Quality control, (2) Alignment, (3) Quantification, (4) Differential expression and normalization, (5) Visualization, and (6) Pathway analysis.
Figure Legend Snippet: Bioinformatics workflow The workflow shows the bioinformatics pipeline used to analyze the miRNA data generated from Illumina MiSeqDx. The pipeline consists of six components: (1) Quality control, (2) Alignment, (3) Quantification, (4) Differential expression and normalization, (5) Visualization, and (6) Pathway analysis.

Techniques Used: Generated, Expressing

11) Product Images from "Preliminary Investigation of Biogenic Amines in Type I Sourdoughs Produced at Home and Bakery Level"

Article Title: Preliminary Investigation of Biogenic Amines in Type I Sourdoughs Produced at Home and Bakery Level

Journal: Toxins

doi: 10.3390/toxins14050293

Relative abundances (%) of bacterial taxa identified by MiSeq Illumina in sourdough samples. Abbreviations: BL, bakery-level sourdough; HM, home-made sourdough.
Figure Legend Snippet: Relative abundances (%) of bacterial taxa identified by MiSeq Illumina in sourdough samples. Abbreviations: BL, bakery-level sourdough; HM, home-made sourdough.

Techniques Used:

12) Product Images from "Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott–Aldrich syndrome"

Article Title: Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott–Aldrich syndrome

Journal: Nature Medicine

doi: 10.1038/s41591-021-01641-x

Integration site (IS) analyses. Panel A, The number of unique ISs detected in PBMCs at various points (in months) after gene therapy. Most of the samples were analyzed by sonic shearing in Illumina sequencing experiments (triangle), except that the early samples were analyzed using restriction endonucleases in 454 sequencing experiments (circle). Panel B, The Pielou diversity index for the IS population in PBMCs for the different patients. Panel C, A stacked bar representation showing the relative clone size (sonic abundance) of the TOP15 integration site clones detected over time for each patient.
Figure Legend Snippet: Integration site (IS) analyses. Panel A, The number of unique ISs detected in PBMCs at various points (in months) after gene therapy. Most of the samples were analyzed by sonic shearing in Illumina sequencing experiments (triangle), except that the early samples were analyzed using restriction endonucleases in 454 sequencing experiments (circle). Panel B, The Pielou diversity index for the IS population in PBMCs for the different patients. Panel C, A stacked bar representation showing the relative clone size (sonic abundance) of the TOP15 integration site clones detected over time for each patient.

Techniques Used: Sequencing, Clone Assay

13) Product Images from "Resequencing the Escherichia coli genome by GenoCare single molecule sequencing platform"

Article Title: Resequencing the Escherichia coli genome by GenoCare single molecule sequencing platform

Journal: bioRxiv

doi: 10.1101/163089

Fraction of genome covered by depth for GenoCare (left) and Illumina (right) platforms.
Figure Legend Snippet: Fraction of genome covered by depth for GenoCare (left) and Illumina (right) platforms.

Techniques Used:

Depth distribution of GenoCare (top) and Illumina (bottom) reads by genome position, binned by 10kb.
Figure Legend Snippet: Depth distribution of GenoCare (top) and Illumina (bottom) reads by genome position, binned by 10kb.

Techniques Used:

14) Product Images from "HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument"

Article Title: HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01490-15

Base calling differences for the Illumina MiSeq and ABI 3730xl Sanger sequencing methods. A change matrix displays the frequency of nucleotides detected by Sanger (rows) and MiSeq (columns) sequencing. Overall, 832 clinical samples and 11 technical replicate
Figure Legend Snippet: Base calling differences for the Illumina MiSeq and ABI 3730xl Sanger sequencing methods. A change matrix displays the frequency of nucleotides detected by Sanger (rows) and MiSeq (columns) sequencing. Overall, 832 clinical samples and 11 technical replicate

Techniques Used: Sequencing

15) Product Images from "In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain"

Article Title: In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain

Journal: bioRxiv

doi: 10.1101/2021.11.23.469762

A genome-wide screen identifies 300 gene knockouts that boost old neural stem cell activation. a, Screen design overview. Three independent screens were performed with primary NSC cultures from young and old Cas9 mice, each derived from a pool of 6 young (3-4 months old) or 6 old (18-21 months old) Cas9 mice (3 male and 3 female littermates). The active NSC cultures are expanded and then put into a state of quiescence with addition of Bone morphogenetic protein (BMP4) growth factor and removal of EGF growth factor. Quiescent NSCs are infected with a lentiviral genome-wide library of single guide RNAs (sgRNAs) targeting 23,000 genes (with 10 sgRNAs per gene and 15000 control sgRNAs, for a total of 245,000 sgRNAs). Quiescent NSCs are then transitioned from quiescence to activation by removal of BMP4 and addition of EGF growth factor, and then activated NSCs are collected after 4 days (Day 4) by intracellular fluorescence-activated cell sorting (FACS) with a proliferation marker (Ki67) or expanded for 14 days (Day 14). Genomic DNA is prepared and sgRNA constructs are PCR amplified rom Day 4 or Day 14 prior to sequencing on an Illumina Novaseq S4 system. b, Example of intracellular FACS analysis with the proliferation marker Ki67 on NSCs from young (3-4 months old, blue) and old (18-21 months old, red) Cas9 mice. Activated NSCs (steady state, top rows), quiescent NSCs (middle rows), or NSCs that transitioned from quiescence to activation (Day 4) that were used for the genome-wide knockout screens (bottom rows). c, Quantification of NSC activation efficiency at Day 4 assessed by intracellular Ki67 FACS analysis. Dot plot showing mean +/- SEM of the percentage of cells expressing Ki67. Each dot represents an independent primary NSC culture derived from a pool of six young (3–4 months old) or old (18-21 months old) Cas9 mice (3 male and 3 female littermates). P -values determined by two tailed Mann-Whitney test. d, Principal Component Analysis (PCA) performed on all gene scores of each of the three independent screens at Day 14: Young 1, 2, 3 (blue) and Old 1, 2, 3 (red). e , Quantification of NSC activation efficiency at Day 4 as assessed by FACS with Ki67 (proliferation marker). qNSCs were infected with lentivirus targeting control sgRNAs (100 sgRNAs targeting unannotated genomic regions) or a pool of sgRNAs targeted the top 10 genes (5 sgRNAs per gene) from screens 1 and 2 (FDR
Figure Legend Snippet: A genome-wide screen identifies 300 gene knockouts that boost old neural stem cell activation. a, Screen design overview. Three independent screens were performed with primary NSC cultures from young and old Cas9 mice, each derived from a pool of 6 young (3-4 months old) or 6 old (18-21 months old) Cas9 mice (3 male and 3 female littermates). The active NSC cultures are expanded and then put into a state of quiescence with addition of Bone morphogenetic protein (BMP4) growth factor and removal of EGF growth factor. Quiescent NSCs are infected with a lentiviral genome-wide library of single guide RNAs (sgRNAs) targeting 23,000 genes (with 10 sgRNAs per gene and 15000 control sgRNAs, for a total of 245,000 sgRNAs). Quiescent NSCs are then transitioned from quiescence to activation by removal of BMP4 and addition of EGF growth factor, and then activated NSCs are collected after 4 days (Day 4) by intracellular fluorescence-activated cell sorting (FACS) with a proliferation marker (Ki67) or expanded for 14 days (Day 14). Genomic DNA is prepared and sgRNA constructs are PCR amplified rom Day 4 or Day 14 prior to sequencing on an Illumina Novaseq S4 system. b, Example of intracellular FACS analysis with the proliferation marker Ki67 on NSCs from young (3-4 months old, blue) and old (18-21 months old, red) Cas9 mice. Activated NSCs (steady state, top rows), quiescent NSCs (middle rows), or NSCs that transitioned from quiescence to activation (Day 4) that were used for the genome-wide knockout screens (bottom rows). c, Quantification of NSC activation efficiency at Day 4 assessed by intracellular Ki67 FACS analysis. Dot plot showing mean +/- SEM of the percentage of cells expressing Ki67. Each dot represents an independent primary NSC culture derived from a pool of six young (3–4 months old) or old (18-21 months old) Cas9 mice (3 male and 3 female littermates). P -values determined by two tailed Mann-Whitney test. d, Principal Component Analysis (PCA) performed on all gene scores of each of the three independent screens at Day 14: Young 1, 2, 3 (blue) and Old 1, 2, 3 (red). e , Quantification of NSC activation efficiency at Day 4 as assessed by FACS with Ki67 (proliferation marker). qNSCs were infected with lentivirus targeting control sgRNAs (100 sgRNAs targeting unannotated genomic regions) or a pool of sgRNAs targeted the top 10 genes (5 sgRNAs per gene) from screens 1 and 2 (FDR

Techniques Used: Genome Wide, Activation Assay, Mouse Assay, Derivative Assay, Infection, Fluorescence, FACS, Marker, Construct, Polymerase Chain Reaction, Amplification, Sequencing, Knock-Out, Expressing, Two Tailed Test, MANN-WHITNEY

16) Product Images from "Indigo Naturalis Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Modulating the Intestinal Microbiota Community"

Article Title: Indigo Naturalis Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Modulating the Intestinal Microbiota Community

Journal: Molecules

doi: 10.3390/molecules24224086

Evaluation of Illumina MiSeq sequencing data, showing that 12 samples and the selected sequencing depth were sufficient to obtain reliable results. ( A ) Specaccum species accumulation curves determined at the 97% similarity level. ( B ) Rank-Abundance curves of the 12 samples. ( C ) Petal pattern of operational taxonomic units (OTUs). ( D ) Venn diagram indicating different numbers of OTUs in the four groups. Note: C = control group; M = DSS model group; P = SASP group at 125 mg/kg); Q = IN group at 200 mg/kg, Here, n = 3 for each group.
Figure Legend Snippet: Evaluation of Illumina MiSeq sequencing data, showing that 12 samples and the selected sequencing depth were sufficient to obtain reliable results. ( A ) Specaccum species accumulation curves determined at the 97% similarity level. ( B ) Rank-Abundance curves of the 12 samples. ( C ) Petal pattern of operational taxonomic units (OTUs). ( D ) Venn diagram indicating different numbers of OTUs in the four groups. Note: C = control group; M = DSS model group; P = SASP group at 125 mg/kg); Q = IN group at 200 mg/kg, Here, n = 3 for each group.

Techniques Used: Sequencing

17) Product Images from "Epstein–Barr virus-associated gastric cancer reveals intratumoral heterogeneity of PIK3CA mutations"

Article Title: Epstein–Barr virus-associated gastric cancer reveals intratumoral heterogeneity of PIK3CA mutations

Journal: Annals of Oncology

doi: 10.1093/annonc/mdx047

Study design. In a first step, the entire GC cohort (n = 484) was screened for EBV association and the presence of PIK3CA hot spot mutations of exon 9 and exon 20 using pyrosequencing. After validation of the significant enrichment of PIK3CA mutations in EBVaGCs, an extended PIK3CA mutational analysis was carried out for these cases in order to detect additional non-hot spot mutations. For 8 cases, only formalin fixed and paraffin embedded specimens were available, and Sanger sequencing of exon 1 and 9 and extended pyrosequencing of exon 20 was carried out. For 14 cases with available frozen, unfixed specimens (‘frozen’), an additional mutational analysis was done using the Illumina TruSight Tumor 26 panel on the Illumina MiSeq System.
Figure Legend Snippet: Study design. In a first step, the entire GC cohort (n = 484) was screened for EBV association and the presence of PIK3CA hot spot mutations of exon 9 and exon 20 using pyrosequencing. After validation of the significant enrichment of PIK3CA mutations in EBVaGCs, an extended PIK3CA mutational analysis was carried out for these cases in order to detect additional non-hot spot mutations. For 8 cases, only formalin fixed and paraffin embedded specimens were available, and Sanger sequencing of exon 1 and 9 and extended pyrosequencing of exon 20 was carried out. For 14 cases with available frozen, unfixed specimens (‘frozen’), an additional mutational analysis was done using the Illumina TruSight Tumor 26 panel on the Illumina MiSeq System.

Techniques Used: Sequencing

18) Product Images from "Bacterial Complexity of Traditional Mountain Butter Is Affected by the Malga-Farm of Production"

Article Title: Bacterial Complexity of Traditional Mountain Butter Is Affected by the Malga-Farm of Production

Journal: Microorganisms

doi: 10.3390/microorganisms10010017

Relative abundances (%) of the main taxa groups (genus level or above) of bacterial sequences from different TMB samples using Illumina MiSeq. Each bar is the mean value of 3 samples collected in different Mfarms located in the Trentino province (for the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Figure Legend Snippet: Relative abundances (%) of the main taxa groups (genus level or above) of bacterial sequences from different TMB samples using Illumina MiSeq. Each bar is the mean value of 3 samples collected in different Mfarms located in the Trentino province (for the interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Techniques Used:

19) Product Images from "Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion"

Article Title: Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1704766114

Library diversity, directed evolution, and enrichment of distinct antigenic footprints. ( A–D ) Parental and evolved libraries were subjected to high-throughput sequencing using the Illumina MiSeq platform. Following analysis with a custom Perl script, enriched amino acid sequences were plotted in R for both the parental and evolved libraries of region IV ( A ), region V ( B ), region VIII ( C ), and combined regions V + VIII ( D ). Each bubble represents a distinct capsid amino acid sequence with the area proportional to the number of reads for that variant in the respective library. ( E–H ) Amino acid sequence representation was calculated for the 10 variants with the highest representation in each library after subjection to evolution. Percentages represent the number of reads for the variant in the evolved library normalized to the total number of reads containing the antigenic region of interest. “Other” sequences represent all other evolved library amino acid sequences not contained in the top-10 hits.
Figure Legend Snippet: Library diversity, directed evolution, and enrichment of distinct antigenic footprints. ( A–D ) Parental and evolved libraries were subjected to high-throughput sequencing using the Illumina MiSeq platform. Following analysis with a custom Perl script, enriched amino acid sequences were plotted in R for both the parental and evolved libraries of region IV ( A ), region V ( B ), region VIII ( C ), and combined regions V + VIII ( D ). Each bubble represents a distinct capsid amino acid sequence with the area proportional to the number of reads for that variant in the respective library. ( E–H ) Amino acid sequence representation was calculated for the 10 variants with the highest representation in each library after subjection to evolution. Percentages represent the number of reads for the variant in the evolved library normalized to the total number of reads containing the antigenic region of interest. “Other” sequences represent all other evolved library amino acid sequences not contained in the top-10 hits.

Techniques Used: Next-Generation Sequencing, Sequencing, Variant Assay

20) Product Images from "Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons"

Article Title: Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005572

PR8-induced IFN-Is alter the fecal microbiota composition, Analysis of fecal microbiota in WT and Ifnar1 -/- mice performed by MiSeq and 16S qPCR during influenza infection. A) Experimental model. Fecal samples were collected from mice on day 0 before infection and on day 9 after mock and PR8 infection. Mice were euthanized at 17 dpi. B, C) The fecal microbiota from WT and Ifnar1 -/- mice on day 0 before infection (n = 6 WT, n = 6 Ifnar1 -/- ), and on day 9 after mock (n = 3 WT, n = 3 Ifnar1 -/- ) and PR8 infection (n = 3 WT, n = 3 Ifnar1 -/- ) was analyzed by sequencing using the Illumina MiSeq system. Graphed is the average relative abundance of each bacterial phylum (B) and genus (C); the cut-off abundance level was set at 0.5%. D) Analysis of the fecal Enterobacteriaceae using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 Ifnar1 -/- ) and on day 9 after mock (n = 5 WT, n = 4 Ifnar1 -/- ) and PR8 infection (n = 5 WT, n = 4 Ifnar1 -/- ). Copy numbers of Enterobacteriaceae per μl of fecal microbial DNA is shown. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by One-Way ANOVA (Bonferroni multiple comparison test). ***p
Figure Legend Snippet: PR8-induced IFN-Is alter the fecal microbiota composition, Analysis of fecal microbiota in WT and Ifnar1 -/- mice performed by MiSeq and 16S qPCR during influenza infection. A) Experimental model. Fecal samples were collected from mice on day 0 before infection and on day 9 after mock and PR8 infection. Mice were euthanized at 17 dpi. B, C) The fecal microbiota from WT and Ifnar1 -/- mice on day 0 before infection (n = 6 WT, n = 6 Ifnar1 -/- ), and on day 9 after mock (n = 3 WT, n = 3 Ifnar1 -/- ) and PR8 infection (n = 3 WT, n = 3 Ifnar1 -/- ) was analyzed by sequencing using the Illumina MiSeq system. Graphed is the average relative abundance of each bacterial phylum (B) and genus (C); the cut-off abundance level was set at 0.5%. D) Analysis of the fecal Enterobacteriaceae using 16S qPCR. Fecal samples were collected from mice on day 0 before infection (n = 10 WT, n = 8 Ifnar1 -/- ) and on day 9 after mock (n = 5 WT, n = 4 Ifnar1 -/- ) and PR8 infection (n = 5 WT, n = 4 Ifnar1 -/- ). Copy numbers of Enterobacteriaceae per μl of fecal microbial DNA is shown. Each dot represents one mouse, the geometric mean is indicated. P values were calculated by One-Way ANOVA (Bonferroni multiple comparison test). ***p

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Sequencing

21) Product Images from "Inflammation-associated alterations to the intestinal microbiota reduce colonization resistance against non-typhoidal Salmonella during concurrent malaria parasite infection"

Article Title: Inflammation-associated alterations to the intestinal microbiota reduce colonization resistance against non-typhoidal Salmonella during concurrent malaria parasite infection

Journal: Scientific Reports

doi: 10.1038/srep14603

Changes in microbial communities after P. yoelii infection. Illumina MiSeq analysis of 16S rRNA amplicons in fecal DNA extracts from P. yoelii -infected mice. ( A ) Average abundance of microbial communities at the phylum level as determined by percent OTU readings (n = 4) at days 0, 10 and 15. OTU with significant changes are shown in Table 1 . Results for individual animals and mock-treated controls are shown in Fig. S2 . ( B ) Abundance of Turicibacter and Ruminococcus (genus) before and after parasite inoculation. Each line represents individual mice (1–4) with two mice succumbing to morbidity after day 15. ( C ) Alpha diversity of 16S rRNA sequences at different time points after P. yoelii infection, determined using Explicet 46 . ( D ) Principal component analysis at the genus level, plotted using METAGENassist 44 . Groups of mice were co-housed.
Figure Legend Snippet: Changes in microbial communities after P. yoelii infection. Illumina MiSeq analysis of 16S rRNA amplicons in fecal DNA extracts from P. yoelii -infected mice. ( A ) Average abundance of microbial communities at the phylum level as determined by percent OTU readings (n = 4) at days 0, 10 and 15. OTU with significant changes are shown in Table 1 . Results for individual animals and mock-treated controls are shown in Fig. S2 . ( B ) Abundance of Turicibacter and Ruminococcus (genus) before and after parasite inoculation. Each line represents individual mice (1–4) with two mice succumbing to morbidity after day 15. ( C ) Alpha diversity of 16S rRNA sequences at different time points after P. yoelii infection, determined using Explicet 46 . ( D ) Principal component analysis at the genus level, plotted using METAGENassist 44 . Groups of mice were co-housed.

Techniques Used: Infection, Mouse Assay

22) Product Images from "Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation"

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

Journal: Synthetic and Systems Biotechnology

doi: 10.1016/j.synbio.2019.01.002

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

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

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

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

23) Product Images from "High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni"

Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

Journal: mBio

doi: 10.1128/mBio.00612-15

Identification of genetic heterogeneity within single colonies and other C. jejuni type strains, and effects of stress selection on mutant populations. Each heat map depicts the resistant/sensitive stress phenotypes, color coded as indicated by the key in panel A, of 96 progeny derived from the indicated strains and conditions, with a list of alleles detected by amplicon sequencing and their relative levels of abundance (maximum = 1.0 [100%]; the frequency listed is the ratio of allele to reference sequence at the specific base position) below. Alleles with synonymous mutations are indicated with a red “S”. Each circle in a heat map represents the average of the spot growth densitometry measurements from three independent experiments performed with OD-standardized cultures, with the average densitometry value being relative to the average value for all colony isolates grown on MH agar alone under standard conditions. (A) Stress phenotype variation and heterogeneity of purF and apt alleles within single-colony-derived representative isolates. The original Sanger sequence genotype of the representative variant colony is listed above the phenotypic heat map, with sequence variants detected by Illumina MiSeq-based amplicon sequencing listed below the heat map. (B) Stress phenotype analysis of 96 colonies derived from parental C. jejuni type strains 81116, NCTC11168-GS, NCTC11168-O, and 81-176, and heterogeneity of purF and apt alleles of each strain. Sequence variant analysis was performed using the known reference sequence specific to each type strain. (C) Effects of exposure to atmospheric O 2 or repeated treatment with MH broth containing 1.0% NaCl on progeny phenotypes and purF and apt genotypes. For O 2 exposure, the parental 81-176 strain was exposed to atmosphere for 48 h and then plated for single colonies on MH agar, from which 96 progeny were derived. The resistant/sensitive phenotype of each of the 96 progeny was retested by exposure to atmospheric O 2 for 48 h prior to phenotypic assessment on MH plates. Genomic DNA for amplicon sequencing was obtained from pooled viable colonies after the initial O 2 exposure. For successive NaCl exposure, strain 81-176 was grown in MH broth or MH broth with 1.0% NaCl for 24 h, and then NaCl-treated cells were used for repeated treatment. At each treatment endpoint, cultures were plated on MH agar to obtain viable single colonies. Stress phenotypes for 96 random colonies from MH agar were obtained by subsequent testing on MH agar with 1.0% NaCl. Genotypes of purF and apt were assessed by amplicon sequencing of genomic DNA from pooled viable colonies from MH agar.
Figure Legend Snippet: Identification of genetic heterogeneity within single colonies and other C. jejuni type strains, and effects of stress selection on mutant populations. Each heat map depicts the resistant/sensitive stress phenotypes, color coded as indicated by the key in panel A, of 96 progeny derived from the indicated strains and conditions, with a list of alleles detected by amplicon sequencing and their relative levels of abundance (maximum = 1.0 [100%]; the frequency listed is the ratio of allele to reference sequence at the specific base position) below. Alleles with synonymous mutations are indicated with a red “S”. Each circle in a heat map represents the average of the spot growth densitometry measurements from three independent experiments performed with OD-standardized cultures, with the average densitometry value being relative to the average value for all colony isolates grown on MH agar alone under standard conditions. (A) Stress phenotype variation and heterogeneity of purF and apt alleles within single-colony-derived representative isolates. The original Sanger sequence genotype of the representative variant colony is listed above the phenotypic heat map, with sequence variants detected by Illumina MiSeq-based amplicon sequencing listed below the heat map. (B) Stress phenotype analysis of 96 colonies derived from parental C. jejuni type strains 81116, NCTC11168-GS, NCTC11168-O, and 81-176, and heterogeneity of purF and apt alleles of each strain. Sequence variant analysis was performed using the known reference sequence specific to each type strain. (C) Effects of exposure to atmospheric O 2 or repeated treatment with MH broth containing 1.0% NaCl on progeny phenotypes and purF and apt genotypes. For O 2 exposure, the parental 81-176 strain was exposed to atmosphere for 48 h and then plated for single colonies on MH agar, from which 96 progeny were derived. The resistant/sensitive phenotype of each of the 96 progeny was retested by exposure to atmospheric O 2 for 48 h prior to phenotypic assessment on MH plates. Genomic DNA for amplicon sequencing was obtained from pooled viable colonies after the initial O 2 exposure. For successive NaCl exposure, strain 81-176 was grown in MH broth or MH broth with 1.0% NaCl for 24 h, and then NaCl-treated cells were used for repeated treatment. At each treatment endpoint, cultures were plated on MH agar to obtain viable single colonies. Stress phenotypes for 96 random colonies from MH agar were obtained by subsequent testing on MH agar with 1.0% NaCl. Genotypes of purF and apt were assessed by amplicon sequencing of genomic DNA from pooled viable colonies from MH agar.

Techniques Used: Selection, Mutagenesis, Derivative Assay, Amplification, Sequencing, Variant Assay

24) Product Images from "Acceleration and suppression of resistance development by antibiotic combinations"

Article Title: Acceleration and suppression of resistance development by antibiotic combinations

Journal: BMC Genomics

doi: 10.1186/s12864-017-3718-2

The number of fixed mutations in evolved strains. Mutations were identified using Illumina MiSeq system (see Methods for details). Blue and green bars represent single nucleotide variation (SNV) and ins/del, respectively
Figure Legend Snippet: The number of fixed mutations in evolved strains. Mutations were identified using Illumina MiSeq system (see Methods for details). Blue and green bars represent single nucleotide variation (SNV) and ins/del, respectively

Techniques Used:

25) Product Images from "5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine"

Article Title: 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.03084-15

5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC 75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant ( P > 0.05).
Figure Legend Snippet: 5-Azacytidine and 5-aza-2′-deoxycytidine induce similar levels of G-to-C and C-to-G transversion mutations during HIV-1 replication. In order to determine whether 5-azacytidine (5-aza-C) and 5-aza-2′-deoxycytidine (5-aza-dC) induce similar changes in HIV-1 mutation frequencies and spectra, U373-MAGI cells were treated with DMSO (no-drug control), 5-aza-C, or 5-aza-dC. 5-Aza-C and 5-aza-dC were added 2 h before infection at the EC 75 (∼260 or 3.8 μM, respectively). Cells were infected at an MOI of 1.0 with NL4-3 MIG-VSVG and were collected 72 h postinfection for the purification of genomic DNA. PCR was performed to prepare multiple amplicons (Gag, Pol, Vif, Env, Nef) from proviral DNA; these were then pooled, used to prepare libraries, and analyzed by 2 × 250 paired-end sequencing on the Illumina MiSeq system. Plasmid control amplifications were performed to determine the levels of background errors resulting from PCR and sequencing. Mutation frequencies for each amplicon, expressed as the number of mutations per base pair, were calculated by dividing the number of mutations by the number of reference bases (mutations + wild-type bases). Data represent means ± standard deviations for three independent biological replicates; N.S., not significant ( P > 0.05).

Techniques Used: Mutagenesis, Infection, Purification, Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Amplification

26) Product Images from "Long-term Proton Pump Inhibitor Administration Caused Physiological and Microbiota Changes in Rats"

Article Title: Long-term Proton Pump Inhibitor Administration Caused Physiological and Microbiota Changes in Rats

Journal: Scientific Reports

doi: 10.1038/s41598-020-57612-8

Fecal microbiome distribution in rats with long-term omeprazole treatment. The feces of rats treated with and without omeprazole for 30 days were prepared for fecal microbiome profiling by high-throughput sequencing of the 16s rRNA gene with the Illumina MiSeq system. ( a ) Alpha-diversity of omeprazole treated samples and untreated controls. Statistical comparison between two groups was performed with exact Wilcoxon-Mann-Whitney test and significant differences were obtained for all four indices (at α = 0.05) ( b ) Principal coordinate analysis (PCoA) plot based on Unweighted or Weight UniFrac distance of omeprazole treated samples and untreated controls. Significant difference in beta-diversity was evaluated with permutational multivariate analysis of variance (vegan::adonis, 1000 permutations) and beta-dispersion was quantified with betadisper (vegan::betadisper, 1000 permutations). Both indices achieved adonis P
Figure Legend Snippet: Fecal microbiome distribution in rats with long-term omeprazole treatment. The feces of rats treated with and without omeprazole for 30 days were prepared for fecal microbiome profiling by high-throughput sequencing of the 16s rRNA gene with the Illumina MiSeq system. ( a ) Alpha-diversity of omeprazole treated samples and untreated controls. Statistical comparison between two groups was performed with exact Wilcoxon-Mann-Whitney test and significant differences were obtained for all four indices (at α = 0.05) ( b ) Principal coordinate analysis (PCoA) plot based on Unweighted or Weight UniFrac distance of omeprazole treated samples and untreated controls. Significant difference in beta-diversity was evaluated with permutational multivariate analysis of variance (vegan::adonis, 1000 permutations) and beta-dispersion was quantified with betadisper (vegan::betadisper, 1000 permutations). Both indices achieved adonis P

Techniques Used: Next-Generation Sequencing, MANN-WHITNEY

27) Product Images from "Tracking the evolution of epialleles during neural differentiation and brain development: D-Aspartate oxidase as a model gene"

Article Title: Tracking the evolution of epialleles during neural differentiation and brain development: D-Aspartate oxidase as a model gene

Journal: Epigenetics

doi: 10.1080/15592294.2016.1260211

Quantitative methylation analysis of a 3 kb genomic region of Ddo gene. A) Structure of the putative mouse Ddo gene promoter. Blue arrow indicates the transcription start site (+1). White box represents the putative regulatory upstream region. Black box represents exon 1. Gray box represents first intron. Positions of CpG sites are indicated as relative to TSS. The analyzed 3 kb genomic portion is divided in 7 regions (R1-R2-R3-R4-R5-R6-R7) corresponding to different amplicons. B) Graph represents methylation average at each CpG site analyzed by Illumina MiSeq Sequencer. Each color line represents the average of 3 mice at a given developmental stage. Blue = E15; red = P0; green = P30. Error bars show standard deviations. For each CpG site, the points labeled with different letters on top are significantly different ( P
Figure Legend Snippet: Quantitative methylation analysis of a 3 kb genomic region of Ddo gene. A) Structure of the putative mouse Ddo gene promoter. Blue arrow indicates the transcription start site (+1). White box represents the putative regulatory upstream region. Black box represents exon 1. Gray box represents first intron. Positions of CpG sites are indicated as relative to TSS. The analyzed 3 kb genomic portion is divided in 7 regions (R1-R2-R3-R4-R5-R6-R7) corresponding to different amplicons. B) Graph represents methylation average at each CpG site analyzed by Illumina MiSeq Sequencer. Each color line represents the average of 3 mice at a given developmental stage. Blue = E15; red = P0; green = P30. Error bars show standard deviations. For each CpG site, the points labeled with different letters on top are significantly different ( P

Techniques Used: Methylation, Mouse Assay, Labeling

28) Product Images from "Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens"

Article Title: Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens

Journal: Nature Microbiology

doi: 10.1038/s41564-019-0626-z

Comparison of taxonomic profiles using Illumina and MinION technology for preterm P8. ( a ) Taxonomic profiles obtained using Illumina HiSeq 2500 and MinION technology for preterm P8. Figure legend comprises the 15 most abundant taxa classified. ( b ) Species-level correlation plot between normalised read count using Illumina (x-axis) and MinION (y-axis) sequencing. Taxa n=121, with log transformed Pearson’s r = 0.91. The grey region either side of fit line represents 95% confidence intervals.
Figure Legend Snippet: Comparison of taxonomic profiles using Illumina and MinION technology for preterm P8. ( a ) Taxonomic profiles obtained using Illumina HiSeq 2500 and MinION technology for preterm P8. Figure legend comprises the 15 most abundant taxa classified. ( b ) Species-level correlation plot between normalised read count using Illumina (x-axis) and MinION (y-axis) sequencing. Taxa n=121, with log transformed Pearson’s r = 0.91. The grey region either side of fit line represents 95% confidence intervals.

Techniques Used: Sequencing, Transformation Assay

Benchmarking and validation of MinION and NanoOK RT using mock resistome samples. a , A known mock community comprising eight bacteria and an isolate of K. pneumoniae from P8 was sequenced using MinION and analysed with NanoOK RT. Findings were compared to the results obtained from the P8 K. pneumoniae isolate sequenced and assembled by Illumina and MinION. The AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. Details of specific genes detected can be found in Supplementary Table 9 . b , MinION AMR profiles obtained from a faecal sample from a healthy preterm infant (P103M no spike) were compared to the K. pneumoniae spiked forms. Two different concentrations of P8 K. pneumoniae isolate were inoculated on sample P103M; 4% (P103M 4% spike) and 40% (P103M 40% spike). Findings were compared to the results obtained from the P8 K. pneumoniae isolate sequenced and assembled from Illumina and MinION. AMR genes were grouped according to sequence similarity. c , Percentages of reads assigned to P103M spiked with 40% P8 K. pneumoniae isolate (P103M 40% spike) and 4% P8 K. pneumoniae isolate (P103M 4% spike); only taxa representing ≥ 1% are shown.
Figure Legend Snippet: Benchmarking and validation of MinION and NanoOK RT using mock resistome samples. a , A known mock community comprising eight bacteria and an isolate of K. pneumoniae from P8 was sequenced using MinION and analysed with NanoOK RT. Findings were compared to the results obtained from the P8 K. pneumoniae isolate sequenced and assembled by Illumina and MinION. The AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. Details of specific genes detected can be found in Supplementary Table 9 . b , MinION AMR profiles obtained from a faecal sample from a healthy preterm infant (P103M no spike) were compared to the K. pneumoniae spiked forms. Two different concentrations of P8 K. pneumoniae isolate were inoculated on sample P103M; 4% (P103M 4% spike) and 40% (P103M 40% spike). Findings were compared to the results obtained from the P8 K. pneumoniae isolate sequenced and assembled from Illumina and MinION. AMR genes were grouped according to sequence similarity. c , Percentages of reads assigned to P103M spiked with 40% P8 K. pneumoniae isolate (P103M 40% spike) and 4% P8 K. pneumoniae isolate (P103M 4% spike); only taxa representing ≥ 1% are shown.

Techniques Used: Sequencing

AMR genes associated to Klebsiella pneumoniae from a metagenomic sample P8 compared to those found in P8 Klebsiella pneumoniae isolate. Heat maps displaying AMR genes associated to K. pneumoniae from faecal sample P8 and AMR genes detected from a K. pneumoniae isolate from the same sample. Sequencing of the metagenomic sample was performed for only 6h using MinION and NanoOK RT tool (highlighted as ‘P8’’). Sequencing and assembly of the K. pneumoniae isolate was performed using both MinION (‘Isolate MinION’) and Illumina HiSeq (‘Isolate Illumina’’). AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. The ”Walkout taxa” column shows the taxa that NanoOK RT’s walkout function classified the nanopore AMR hits to. An “n/a” indicates the gene is not found in the metagenomic nanopore sample. A single read can only be classified to a single taxa, but multiple taxa are shown if different reads classify to different taxa. Further details of specific gene names can be found in Supplementary Table 8 .
Figure Legend Snippet: AMR genes associated to Klebsiella pneumoniae from a metagenomic sample P8 compared to those found in P8 Klebsiella pneumoniae isolate. Heat maps displaying AMR genes associated to K. pneumoniae from faecal sample P8 and AMR genes detected from a K. pneumoniae isolate from the same sample. Sequencing of the metagenomic sample was performed for only 6h using MinION and NanoOK RT tool (highlighted as ‘P8’’). Sequencing and assembly of the K. pneumoniae isolate was performed using both MinION (‘Isolate MinION’) and Illumina HiSeq (‘Isolate Illumina’’). AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. The ”Walkout taxa” column shows the taxa that NanoOK RT’s walkout function classified the nanopore AMR hits to. An “n/a” indicates the gene is not found in the metagenomic nanopore sample. A single read can only be classified to a single taxa, but multiple taxa are shown if different reads classify to different taxa. Further details of specific gene names can be found in Supplementary Table 8 .

Techniques Used: Sequencing

Sequencing of microbial mock community (HM-277D) with Illumina and MinION sequencing. a , Correlation plot comparing the expected proportions of DNA from each of the 20 species in the mock community with the proportions of sequence obtained by MinION (left, log-transformed Pearson’s r = 0.94) and Illumina platforms (log-transformed Pearson’s r = 0.97). The right-hand plot shows the correlation between MinION and Illumina (log-transformed Pearson’s r = 0.98). The grey region either side of the fit line represents the 95% CIs. b , MEGAN 37 taxonomy tree representing taxa assigned from the mock community as sequenced by Illumina MiSeq (green) and ONT MinION (brown). The height of the bars indicates the relative number of reads assigned, with normalized counts given next to the taxa name. Asterisks represent species assigned by MEGAN but not specified as members of the mock community. Numbers assigned to taxa correspond to Illumina MiSeq (left) and ONT MinION (right) normalized counts, respectively.
Figure Legend Snippet: Sequencing of microbial mock community (HM-277D) with Illumina and MinION sequencing. a , Correlation plot comparing the expected proportions of DNA from each of the 20 species in the mock community with the proportions of sequence obtained by MinION (left, log-transformed Pearson’s r = 0.94) and Illumina platforms (log-transformed Pearson’s r = 0.97). The right-hand plot shows the correlation between MinION and Illumina (log-transformed Pearson’s r = 0.98). The grey region either side of the fit line represents the 95% CIs. b , MEGAN 37 taxonomy tree representing taxa assigned from the mock community as sequenced by Illumina MiSeq (green) and ONT MinION (brown). The height of the bars indicates the relative number of reads assigned, with normalized counts given next to the taxa name. Asterisks represent species assigned by MEGAN but not specified as members of the mock community. Numbers assigned to taxa correspond to Illumina MiSeq (left) and ONT MinION (right) normalized counts, respectively.

Techniques Used: Sequencing, Transformation Assay

Longitudinal study on preterm infant P10 using MinION and Illumina sequencing. a , Timeline diagram of preterm P10 indicating the times of faecal sample collection (P10N, P10R and P10V), duration of antibiotic and probiotic treatment and relevant clinical observations. The diagram is divided into weeks and the dots represent days within the timescale. The blue dots represent the days of probiotic treatment; the red dots represent the days of antibiotic treatment; the black squares represent the time points for sample collection; and the letter H represents the transfer of the preterm infant to another hospital. b , Correlation plot of normalized species abundance in taxonomic assignments of Illumina ( x axis) and MinION ( y axis) data for sample P10N, with log-transformed Pearson’s r = 0.95 (taxa n = 92). The values for P10R and P10V were 0.90 and 0.94, respectively. The grey region either side of the fit line represents the 95% CIs. c , Taxonomic profiles at time points P10N, P10R and P10V, as assigned by MEGAN. The top row corresponds to the results obtained using MinION sequencing; the bottom row displays the results obtained using Illumina HiSeq. The legend comprises the 12 most abundant taxa obtained. Further information on all taxa and corresponding read counts can be found in Supplementary Table 3 .
Figure Legend Snippet: Longitudinal study on preterm infant P10 using MinION and Illumina sequencing. a , Timeline diagram of preterm P10 indicating the times of faecal sample collection (P10N, P10R and P10V), duration of antibiotic and probiotic treatment and relevant clinical observations. The diagram is divided into weeks and the dots represent days within the timescale. The blue dots represent the days of probiotic treatment; the red dots represent the days of antibiotic treatment; the black squares represent the time points for sample collection; and the letter H represents the transfer of the preterm infant to another hospital. b , Correlation plot of normalized species abundance in taxonomic assignments of Illumina ( x axis) and MinION ( y axis) data for sample P10N, with log-transformed Pearson’s r = 0.95 (taxa n = 92). The values for P10R and P10V were 0.90 and 0.94, respectively. The grey region either side of the fit line represents the 95% CIs. c , Taxonomic profiles at time points P10N, P10R and P10V, as assigned by MEGAN. The top row corresponds to the results obtained using MinION sequencing; the bottom row displays the results obtained using Illumina HiSeq. The legend comprises the 12 most abundant taxa obtained. Further information on all taxa and corresponding read counts can be found in Supplementary Table 3 .

Techniques Used: Sequencing, Transformation Assay

Presence/absence heat maps of AMR hits found in P10 using MinION and Illumina. Three samples of preterm P10 were analysed and sequenced using MinION (P10N-N, P10R-N and P10V-N), and Illumina technology (P10N-I, P10R-I and P10V-I). ( a ) Heat map representing efflux pumps inhibitors or regulators genes found in samples P10N, P10R and P10V. ( b ) Heat map highlighting b-lactamases. ( c ) Heat map showing (1) aminocoumarin resistance genes (2) aminoglycosides resistance (3) bacitracin resistance (4) colistin resistance (5) erythromycin resistance (6) fluoroquinolone resistance (7) fosfomycin resistance (8) mupirocin resistance (9) quinolone resistance (10) streptothricin resistance (11) sulphonamide resistance (12) tetracycline resistance (13) trimethoprim resistance. AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. Further information on genes detected can be found in Supplementary Table 2 .
Figure Legend Snippet: Presence/absence heat maps of AMR hits found in P10 using MinION and Illumina. Three samples of preterm P10 were analysed and sequenced using MinION (P10N-N, P10R-N and P10V-N), and Illumina technology (P10N-I, P10R-I and P10V-I). ( a ) Heat map representing efflux pumps inhibitors or regulators genes found in samples P10N, P10R and P10V. ( b ) Heat map highlighting b-lactamases. ( c ) Heat map showing (1) aminocoumarin resistance genes (2) aminoglycosides resistance (3) bacitracin resistance (4) colistin resistance (5) erythromycin resistance (6) fluoroquinolone resistance (7) fosfomycin resistance (8) mupirocin resistance (9) quinolone resistance (10) streptothricin resistance (11) sulphonamide resistance (12) tetracycline resistance (13) trimethoprim resistance. AMR genes were grouped according to sequence similarity. Blue indicates presence, grey indicates absence. Further information on genes detected can be found in Supplementary Table 2 .

Techniques Used: Sequencing

29) Product Images from "Specialisation events of fungal metacommunities exposed to a persistent organic pollutant are suggestive of augmented pathogenic potential"

Article Title: Specialisation events of fungal metacommunities exposed to a persistent organic pollutant are suggestive of augmented pathogenic potential

Journal: Microbiome

doi: 10.1186/s40168-018-0589-y

Taxonomic diversity of the metacommunity of fungi in the inoculum. Cladogram based on the ITS2 sequence similarity illustrating the diversity of OTUs identified in the metacommunity inoculum by amplicon sequencing (left) and their relative abundance per taxonomic order, comprising also unknown fungi ( incertae sedis ), using the normalised read counts, sub-sampled for the sequencing depth of the Illumina MiSeq run (100000 reads) (right)
Figure Legend Snippet: Taxonomic diversity of the metacommunity of fungi in the inoculum. Cladogram based on the ITS2 sequence similarity illustrating the diversity of OTUs identified in the metacommunity inoculum by amplicon sequencing (left) and their relative abundance per taxonomic order, comprising also unknown fungi ( incertae sedis ), using the normalised read counts, sub-sampled for the sequencing depth of the Illumina MiSeq run (100000 reads) (right)

Techniques Used: Sequencing, Amplification

30) Product Images from "Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples"

Article Title: Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples

Journal: Oncotarget

doi: 10.18632/oncotarget.11259

Distribution of coverage for each amplicon in BRCA1 a. and BRCA2 b. originating from Illumina MiSeq sequencing data Each amplicon was surveyed at multiple reference points. Average was calculated from 24 samples. Error bars represent the minimum and maximum values of coverage in the respective amplicon.
Figure Legend Snippet: Distribution of coverage for each amplicon in BRCA1 a. and BRCA2 b. originating from Illumina MiSeq sequencing data Each amplicon was surveyed at multiple reference points. Average was calculated from 24 samples. Error bars represent the minimum and maximum values of coverage in the respective amplicon.

Techniques Used: Amplification, Sequencing

31) Product Images from "In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening"

Article Title: In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01524-21

Relative abundances (%) of bacteria identified by MiSeq Illumina from the biofilms on the wooden shelves used to ripen PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheeses in 18 dairy facilities. The amplification occurred on a 464-nucleotide sequence of the V3-V4 region of the 16S rRNA gene. Only taxa occurring at > 0.1% abundance in at least one sample were included. Abbreviations: WSA to -T, wooden shelves from factories A to T, respectively.
Figure Legend Snippet: Relative abundances (%) of bacteria identified by MiSeq Illumina from the biofilms on the wooden shelves used to ripen PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheeses in 18 dairy facilities. The amplification occurred on a 464-nucleotide sequence of the V3-V4 region of the 16S rRNA gene. Only taxa occurring at > 0.1% abundance in at least one sample were included. Abbreviations: WSA to -T, wooden shelves from factories A to T, respectively.

Techniques Used: Amplification, Sequencing

32) Product Images from "Specialisation events of fungal metacommunities exposed to a persistent organic pollutant are suggestive of augmented pathogenic potential"

Article Title: Specialisation events of fungal metacommunities exposed to a persistent organic pollutant are suggestive of augmented pathogenic potential

Journal: Microbiome

doi: 10.1186/s40168-018-0589-y

Taxonomic diversity of the metacommunity of fungi in the inoculum. Cladogram based on the ITS2 sequence similarity illustrating the diversity of OTUs identified in the metacommunity inoculum by amplicon sequencing (left) and their relative abundance per taxonomic order, comprising also unknown fungi ( incertae sedis ), using the normalised read counts, sub-sampled for the sequencing depth of the Illumina MiSeq run (100000 reads) (right)
Figure Legend Snippet: Taxonomic diversity of the metacommunity of fungi in the inoculum. Cladogram based on the ITS2 sequence similarity illustrating the diversity of OTUs identified in the metacommunity inoculum by amplicon sequencing (left) and their relative abundance per taxonomic order, comprising also unknown fungi ( incertae sedis ), using the normalised read counts, sub-sampled for the sequencing depth of the Illumina MiSeq run (100000 reads) (right)

Techniques Used: Sequencing, Amplification

33) Product Images from "Insight into the bacterial communities of the subterranean aphid Anoecia corni"

Article Title: Insight into the bacterial communities of the subterranean aphid Anoecia corni

Journal: PLoS ONE

doi: 10.1371/journal.pone.0256019

Relative abundance of bacterial taxa from Illumina sequencing of 16S rRNA amplicons, represented as a heat map based on the log-transformed values. The warm colors indicating higher and cold colors indicating lower abundance. Each color bar corresponding to one sampled colony.
Figure Legend Snippet: Relative abundance of bacterial taxa from Illumina sequencing of 16S rRNA amplicons, represented as a heat map based on the log-transformed values. The warm colors indicating higher and cold colors indicating lower abundance. Each color bar corresponding to one sampled colony.

Techniques Used: Sequencing, Transformation Assay

34) Product Images from "Genome sequence and transcriptomic profiles of a marine bacterium, Pseudoalteromonas agarivorans Hao 2018"

Article Title: Genome sequence and transcriptomic profiles of a marine bacterium, Pseudoalteromonas agarivorans Hao 2018

Journal: Scientific Data

doi: 10.1038/s41597-019-0012-y

Quality control of the genome sequencing data. ( a ) Base quality using Phred scores for the data from Illumina Miseq sequencing. In the box plots, the whiskers represent the range between the 10 and 90% quantiles; the yellow boxes show the range between the upper and lower quartiles; the red lines represent median. The blue line indicates the mean quality. ( b ) Distribution of read-over-read quality. ( c ) Read length distribution of PacBio sequencing after filtering. ( d ) Read quality distribution of PacBio sequencing after filtering.
Figure Legend Snippet: Quality control of the genome sequencing data. ( a ) Base quality using Phred scores for the data from Illumina Miseq sequencing. In the box plots, the whiskers represent the range between the 10 and 90% quantiles; the yellow boxes show the range between the upper and lower quartiles; the red lines represent median. The blue line indicates the mean quality. ( b ) Distribution of read-over-read quality. ( c ) Read length distribution of PacBio sequencing after filtering. ( d ) Read quality distribution of PacBio sequencing after filtering.

Techniques Used: Sequencing

35) Product Images from "High-Throughput Sequencing of Human Immunoglobulin Variable Regions with Subtype Identification"

Article Title: High-Throughput Sequencing of Human Immunoglobulin Variable Regions with Subtype Identification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111726

Amplification of Ig variable regions and high-throughput sequencing with subtype information. Antibody heavy and light chain genes are shown schematically with leader regions in purple, variable (V-) regions in blue and constant (C-) regions in green. Ig heavy, kappa and lambda light chain genes were amplified in separate reactions with family specific primers (represented by arrows) binding to leader and constant region. Primer names are indicated exemplarily below the arrows and a complete list of all primers used can be found in Table 1 . Sequencing adaptors essential for the Illumina platform (flow cell binding sites P5 and P7, the index 2 region i5 and the read 1 sequencing primer binding site Rd1 SP, illustrated in orange) were added by primer extension during a second PCR reaction, except for the IgG reverse primer TS7IgG(int) which contained an adaptor and was used for both amplifications. Purified libraries were then sequenced using standard Illumina MiSeq primers for read 1 and index 2, and customized primers for index 1 and read 2 (sequencing primers are shown in red and regions sequenced with red dashed arrows).
Figure Legend Snippet: Amplification of Ig variable regions and high-throughput sequencing with subtype information. Antibody heavy and light chain genes are shown schematically with leader regions in purple, variable (V-) regions in blue and constant (C-) regions in green. Ig heavy, kappa and lambda light chain genes were amplified in separate reactions with family specific primers (represented by arrows) binding to leader and constant region. Primer names are indicated exemplarily below the arrows and a complete list of all primers used can be found in Table 1 . Sequencing adaptors essential for the Illumina platform (flow cell binding sites P5 and P7, the index 2 region i5 and the read 1 sequencing primer binding site Rd1 SP, illustrated in orange) were added by primer extension during a second PCR reaction, except for the IgG reverse primer TS7IgG(int) which contained an adaptor and was used for both amplifications. Purified libraries were then sequenced using standard Illumina MiSeq primers for read 1 and index 2, and customized primers for index 1 and read 2 (sequencing primers are shown in red and regions sequenced with red dashed arrows).

Techniques Used: Amplification, Next-Generation Sequencing, Binding Assay, Sequencing, Flow Cytometry, Polymerase Chain Reaction, Purification

36) Product Images from "Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol"

Article Title: Whole-Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina MiSeq ARTIC Protocol

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00649-21

(A) Genome coverage of the 181 primary samples that returned sequences using the AmpliSeq method, compared to the C T value obtained with the CDC 2019-nCoV real-time RT-PCR diagnostic panel as an indication of viral load. The percentage of bases with at least 20× coverage was reported by the CoverageAnalysis plug-in. (B) Genome coverage of the 83 samples that were processed with both the AmpliSeq Ion Torrent and Illumina MiSeq ARTIC methods.
Figure Legend Snippet: (A) Genome coverage of the 181 primary samples that returned sequences using the AmpliSeq method, compared to the C T value obtained with the CDC 2019-nCoV real-time RT-PCR diagnostic panel as an indication of viral load. The percentage of bases with at least 20× coverage was reported by the CoverageAnalysis plug-in. (B) Genome coverage of the 83 samples that were processed with both the AmpliSeq Ion Torrent and Illumina MiSeq ARTIC methods.

Techniques Used: Quantitative RT-PCR, Diagnostic Assay

Workflow for each of the methods described and the associated preparation and incubation times for each step. The ARTIC Illumina workflow is for 94 samples and 2 negative-control samples, and the AmpliSeq workflow is for 64 samples using a single Ion Chef instrument. QC, quality control.
Figure Legend Snippet: Workflow for each of the methods described and the associated preparation and incubation times for each step. The ARTIC Illumina workflow is for 94 samples and 2 negative-control samples, and the AmpliSeq workflow is for 64 samples using a single Ion Chef instrument. QC, quality control.

Techniques Used: Incubation, Negative Control

37) Product Images from "Resequencing the Escherichia coli genome by GenoCare single molecule sequencing platform"

Article Title: Resequencing the Escherichia coli genome by GenoCare single molecule sequencing platform

Journal: bioRxiv

doi: 10.1101/163089

Fraction of genome covered by depth for GenoCare (left) and Illumina (right) platforms.
Figure Legend Snippet: Fraction of genome covered by depth for GenoCare (left) and Illumina (right) platforms.

Techniques Used:

Depth distribution of GenoCare (top) and Illumina (bottom) reads by genome position, binned by 10kb.
Figure Legend Snippet: Depth distribution of GenoCare (top) and Illumina (bottom) reads by genome position, binned by 10kb.

Techniques Used:

38) Product Images from "Maternal Immune Activation Causes Social Behavior Deficits and Hypomyelination in Male Rat Offspring with an Autism-Like Microbiota Profile"

Article Title: Maternal Immune Activation Causes Social Behavior Deficits and Hypomyelination in Male Rat Offspring with an Autism-Like Microbiota Profile

Journal: Brain Sciences

doi: 10.3390/brainsci11081085

Fecal microbiome distribution in male maternal immune activation (MIA) offspring. The feces of prenatal lipopolysaccharide (LPS)-stimulated male offspring (LPS group) and control rats were prepared for fecal microbiome profiling through high-throughput sequencing of the 16s rRNA gene on the Illumina MiSeq system. ( A ) Alpha diversity of MIA offspring (LPS) and controls. ( B ) Principal coordinate analysis plot based on unweighted or weighted UniFrac distance of omeprazole MIA offspring and control samples. A significant difference in beta diversity was evaluated using permutational multivariate analysis of variance (vegan:adonis, 1000 permutations), and beta dispersion was quantified using a betadisper (vegan:betadisper, 1000 permutations). Both indices achieved adonis p
Figure Legend Snippet: Fecal microbiome distribution in male maternal immune activation (MIA) offspring. The feces of prenatal lipopolysaccharide (LPS)-stimulated male offspring (LPS group) and control rats were prepared for fecal microbiome profiling through high-throughput sequencing of the 16s rRNA gene on the Illumina MiSeq system. ( A ) Alpha diversity of MIA offspring (LPS) and controls. ( B ) Principal coordinate analysis plot based on unweighted or weighted UniFrac distance of omeprazole MIA offspring and control samples. A significant difference in beta diversity was evaluated using permutational multivariate analysis of variance (vegan:adonis, 1000 permutations), and beta dispersion was quantified using a betadisper (vegan:betadisper, 1000 permutations). Both indices achieved adonis p

Techniques Used: Activation Assay, Next-Generation Sequencing

39) Product Images from "Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM)"

Article Title: Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM)

Journal: Journal of Animal Science and Biotechnology

doi: 10.1186/s40104-021-00584-0

Relative abundance of the 15 main bacterial families in the bioreactor medium ( a ) and on the mucin beads ( b ) of the MPigut-IVM during the fermentations #6, 7, 8 and 9 which were subjected to a feed deprivation stress of 48 h, as measured by 16S Illumina sequencing
Figure Legend Snippet: Relative abundance of the 15 main bacterial families in the bioreactor medium ( a ) and on the mucin beads ( b ) of the MPigut-IVM during the fermentations #6, 7, 8 and 9 which were subjected to a feed deprivation stress of 48 h, as measured by 16S Illumina sequencing

Techniques Used: Sequencing

Effects of a feed deprivation stress of 12, 24, and 48 h on the MPigut-IVM microbiota composition. On this figure, the “12h feed deprivation stress”, “24h feed deprivation stress” and “48h feed deprivation stress” correspond to the fermentations #4, 5 and 6, respectively. a Evolution of the redox potential. An example of redox measurement for a control assay is provided one the figure. These data are from the fermentation 2. b Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families in the bioreactor medium of the MPigut-IVM as measured by 16S Illumina sequencing. c Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families on the mucin beads of the MPigut-IVM as measured by 16S Illumina sequencing
Figure Legend Snippet: Effects of a feed deprivation stress of 12, 24, and 48 h on the MPigut-IVM microbiota composition. On this figure, the “12h feed deprivation stress”, “24h feed deprivation stress” and “48h feed deprivation stress” correspond to the fermentations #4, 5 and 6, respectively. a Evolution of the redox potential. An example of redox measurement for a control assay is provided one the figure. These data are from the fermentation 2. b Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families in the bioreactor medium of the MPigut-IVM as measured by 16S Illumina sequencing. c Effect of a feed deprivation period of 12, 24 or 48 h on the relative abundance of the main bacterial families on the mucin beads of the MPigut-IVM as measured by 16S Illumina sequencing

Techniques Used: Control Assay, Sequencing

Set-up and validation of the MPigut-IVM: microbiota composition. a Evolution of the structure and colonization of a mucin bead before (I) and after (II) 48 h of incubation in the MPigut-IVM during the fermentation #1 and observation of the specific adherent microbiota at high magnitude (III IV) by scanning electron microscopy. b Relative abundance of the 15 principal bacterial families in the bioreactor medium of the MPigut-IVM during control assays measured by 16S Illumina sequencing. c Relative abundance of the 15 principal bacterial families on the mucin beads of the MPigut-IVM during control assays measured by 16S Illumina sequencing
Figure Legend Snippet: Set-up and validation of the MPigut-IVM: microbiota composition. a Evolution of the structure and colonization of a mucin bead before (I) and after (II) 48 h of incubation in the MPigut-IVM during the fermentation #1 and observation of the specific adherent microbiota at high magnitude (III IV) by scanning electron microscopy. b Relative abundance of the 15 principal bacterial families in the bioreactor medium of the MPigut-IVM during control assays measured by 16S Illumina sequencing. c Relative abundance of the 15 principal bacterial families on the mucin beads of the MPigut-IVM during control assays measured by 16S Illumina sequencing

Techniques Used: Incubation, Electron Microscopy, Sequencing

40) Product Images from "Acceleration and suppression of resistance development by antibiotic combinations"

Article Title: Acceleration and suppression of resistance development by antibiotic combinations

Journal: BMC Genomics

doi: 10.1186/s12864-017-3718-2

The number of fixed mutations in evolved strains. Mutations were identified using Illumina MiSeq system (see Methods for details). Blue and green bars represent single nucleotide variation (SNV) and ins/del, respectively
Figure Legend Snippet: The number of fixed mutations in evolved strains. Mutations were identified using Illumina MiSeq system (see Methods for details). Blue and green bars represent single nucleotide variation (SNV) and ins/del, respectively

Techniques Used:

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    Comparison between <t>Illumina</t> <t>MiSeq</t> (green bars) and Ion Torrent sequencing (gray bars) in reflecting OTU richness (A,B) and evenness (C,D) across the four treatment levels: Lepeophtheirus salmonis (LeSa), Paramoeba perurans (PaPe), algal mix (AlgMix) containing the three algal species, and Mix containing all the five species together. Within each panel the two different methods are compared.
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    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:

    Heat map representing the distribution of samples and OTUs in the T. putrescentiae bacterial community in the standardized Illumina subsample data set. Data were log 2 transformed. The samples are characterized by different colors, as follows: red, Koppert;

    Journal: Applied and Environmental Microbiology

    Article Title: Experimental Manipulation Shows a Greater Influence of Population than Dietary Perturbation on the Microbiome of Tyrophagus putrescentiae

    doi: 10.1128/AEM.00128-17

    Figure Lengend Snippet: Heat map representing the distribution of samples and OTUs in the T. putrescentiae bacterial community in the standardized Illumina subsample data set. Data were log 2 transformed. The samples are characterized by different colors, as follows: red, Koppert;

    Article Snippet: DNA samples were sent to MR DNA (Shallowater, TX, USA) for sequencing of the V1-V3 portions of the 16S rRNA genes using the universal primers 27Fmod and 519Rmod on an Illumina MiSeq platform, employing methods based on the bTEFAP (bacterial tag-encoded FLX amplicon pyrosequencing) process ( ) in MR DNA.

    Techniques: Transformation Assay

    Evaluation of Illumina MiSeq sequencing data, showing that 12 samples and the selected sequencing depth were sufficient to obtain reliable results. ( A ) Specaccum species accumulation curves determined at the 97% similarity level. ( B ) Rank-Abundance curves of the 12 samples. ( C ) Petal pattern of operational taxonomic units (OTUs). ( D ) Venn diagram indicating different numbers of OTUs in the four groups. Note: C = control group; M = DSS model group; P = SASP group at 125 mg/kg); Q = IN group at 200 mg/kg, Here, n = 3 for each group.

    Journal: Molecules

    Article Title: Indigo Naturalis Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Modulating the Intestinal Microbiota Community

    doi: 10.3390/molecules24224086

    Figure Lengend Snippet: Evaluation of Illumina MiSeq sequencing data, showing that 12 samples and the selected sequencing depth were sufficient to obtain reliable results. ( A ) Specaccum species accumulation curves determined at the 97% similarity level. ( B ) Rank-Abundance curves of the 12 samples. ( C ) Petal pattern of operational taxonomic units (OTUs). ( D ) Venn diagram indicating different numbers of OTUs in the four groups. Note: C = control group; M = DSS model group; P = SASP group at 125 mg/kg); Q = IN group at 200 mg/kg, Here, n = 3 for each group.

    Article Snippet: In Ameliorated the Intestine Microecology of Colitis Mice Induced by DSS To determine the effects of IN on the gut microbiota, 16S rDNA gene amplification (V4-V5 region) and sequencing were employed with the Illumina MiSeq platform in our work (see Materials and Methods, ).

    Techniques: Sequencing

    Comparison between Illumina MiSeq (green bars) and Ion Torrent sequencing (gray bars) in reflecting OTU richness (A,B) and evenness (C,D) across the four treatment levels: Lepeophtheirus salmonis (LeSa), Paramoeba perurans (PaPe), algal mix (AlgMix) containing the three algal species, and Mix containing all the five species together. Within each panel the two different methods are compared.

    Journal: Frontiers in Microbiology

    Article Title: Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture

    doi: 10.3389/fmicb.2018.03009

    Figure Lengend Snippet: Comparison between Illumina MiSeq (green bars) and Ion Torrent sequencing (gray bars) in reflecting OTU richness (A,B) and evenness (C,D) across the four treatment levels: Lepeophtheirus salmonis (LeSa), Paramoeba perurans (PaPe), algal mix (AlgMix) containing the three algal species, and Mix containing all the five species together. Within each panel the two different methods are compared.

    Article Snippet: Several hundred samples can be multiplexed on a single Illumina MiSeq run, but the cost per run is relatively high.

    Techniques: Sequencing

    Experimental design aiming to test the efficiency of two sequencing methods, namely Illumina MiSeq and IonTorrent, in reflecting the species composition and abundance in different incubations. Each incubation contained four aquaculture related risk agents either in isolation or combined: Lepeophtheirus salmonis (LeSa), Paramoeba perurans (PaPe), algal mix (AlgMix) containing Prymnesium parvum, Pseudonitzschia delicaticima, and P. seriata , and Mix containing all the species together. Each of these incubations was performed at three abundance levels representing triplings of the initial abundance (i.e., 2, 6, and 18). The experiment aimed to control for the effect of background noise in these treatments thus we deployed these using both filtered and unfiltered seawater medium.

    Journal: Frontiers in Microbiology

    Article Title: Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture

    doi: 10.3389/fmicb.2018.03009

    Figure Lengend Snippet: Experimental design aiming to test the efficiency of two sequencing methods, namely Illumina MiSeq and IonTorrent, in reflecting the species composition and abundance in different incubations. Each incubation contained four aquaculture related risk agents either in isolation or combined: Lepeophtheirus salmonis (LeSa), Paramoeba perurans (PaPe), algal mix (AlgMix) containing Prymnesium parvum, Pseudonitzschia delicaticima, and P. seriata , and Mix containing all the species together. Each of these incubations was performed at three abundance levels representing triplings of the initial abundance (i.e., 2, 6, and 18). The experiment aimed to control for the effect of background noise in these treatments thus we deployed these using both filtered and unfiltered seawater medium.

    Article Snippet: Several hundred samples can be multiplexed on a single Illumina MiSeq run, but the cost per run is relatively high.

    Techniques: Sequencing, Incubation, Isolation