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Illumina Inc single illumina miseq
Percent of reads to bacterial rRNA and host transcripts are increased in depleted samples Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an <t>Illumina</t> <t>MiSeq.</t> Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using either BBSplit to the appropriate reference sequence, 16S bacterial rRNA (A), 23S bacterial rRNA (B), Elizabethkingia anophelis transcriptome (C and D) and the NCBI bacterial genome database (E). The reads called for each gene was determined using BBMap with output flag rpkm (D). All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.
Single Illumina Miseq, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery"

Article Title: A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery

Journal: Virology

doi: 10.1016/j.virol.2018.12.020

Percent of reads to bacterial rRNA and host transcripts are increased in depleted samples Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using either BBSplit to the appropriate reference sequence, 16S bacterial rRNA (A), 23S bacterial rRNA (B), Elizabethkingia anophelis transcriptome (C and D) and the NCBI bacterial genome database (E). The reads called for each gene was determined using BBMap with output flag rpkm (D). All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.
Figure Legend Snippet: Percent of reads to bacterial rRNA and host transcripts are increased in depleted samples Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using either BBSplit to the appropriate reference sequence, 16S bacterial rRNA (A), 23S bacterial rRNA (B), Elizabethkingia anophelis transcriptome (C and D) and the NCBI bacterial genome database (E). The reads called for each gene was determined using BBMap with output flag rpkm (D). All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.

Techniques Used: Purification, Sequencing

Percent of reads to bacterial rRNA and host transcripts are increased in depleted samples. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using either BBMap or BBSplit to the appropriate reference sequence, An. gambiae transcriptome (A and B), West Nile virus (C) or Bolahun virus (D). The reads called for each gene was determined using BBMap with output flag rpkm (B). Variants detected in Bolahun virus were called using LoFreq (F). Only variants present at greater than 5% were used for analysis. All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.
Figure Legend Snippet: Percent of reads to bacterial rRNA and host transcripts are increased in depleted samples. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using either BBMap or BBSplit to the appropriate reference sequence, An. gambiae transcriptome (A and B), West Nile virus (C) or Bolahun virus (D). The reads called for each gene was determined using BBMap with output flag rpkm (B). Variants detected in Bolahun virus were called using LoFreq (F). Only variants present at greater than 5% were used for analysis. All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.

Techniques Used: Purification, Sequencing

Reverse Transcriptase mediated ribosomal RNA (rRNA) depletion increases target-specific coverage while reducing the number of rRNA reads in next-generation sequencing. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNase H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using BBSplit to the appropriate reference sequence, unique reads after duplicate removal (A), 18S rRNA (B), 28S rRNA (C) and mitochondrial 16S rRNA (D). Reads for 18S (E) and 28S (F) rRNA were then normalized to host gene AGAP001826-RA and then compared. All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.
Figure Legend Snippet: Reverse Transcriptase mediated ribosomal RNA (rRNA) depletion increases target-specific coverage while reducing the number of rRNA reads in next-generation sequencing. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted, and DNase treated. This is considered the input RNA. DNase-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNase H and DNase I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Each sample was then normalized to contain 1.5 million reads to allow for direct comparisons. Duplicate reads were removed using Clumpify and then unique reads were mapped using BBSplit to the appropriate reference sequence, unique reads after duplicate removal (A), 18S rRNA (B), 28S rRNA (C) and mitochondrial 16S rRNA (D). Reads for 18S (E) and 28S (F) rRNA were then normalized to host gene AGAP001826-RA and then compared. All statistical tests were performed by One-Way ANOVA with Tukey test for multiple comparisons. The p-values are indicated as numbers. All p-values without an accompanying bar are statistical comparisons between the input RNA and the other group. The other comparisons with a bar are statistical comparisons between the two groups that are below the corresponding bar.

Techniques Used: Next-Generation Sequencing, Purification, Sequencing

2) Product Images from "Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03009

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Sequencing, Incubation, Isolation

Variability in the read numbers of the three target microalgal species namely the two Pseudo-nitzschia species that were assigned as P. cuspidata and P. australis , but which were in fact P. delicatissima and P. seriata (A–D) and Prymnesium parvum (E,F) across the different treatments (see Figure 1 for treatment abbreviations). Within each treatment, the reads of each species were compared between the two sequencing methods Illumina MiSeq and Ion Torrent and between filtered and unfiltered marine plankton samples. For the non-prefiltered seawater samples, increasing relative abundances of reads for the target organisms is evident as their concentration is increased in the original sample for every organism except P. australis . A similar pattern is not observed in prefiltered seawater samples. Low levels of cross contamination between the target species ( P. perurans an L. salmonis reads in algal samples) is also observed.
Figure Legend Snippet: Variability in the read numbers of the three target microalgal species namely the two Pseudo-nitzschia species that were assigned as P. cuspidata and P. australis , but which were in fact P. delicatissima and P. seriata (A–D) and Prymnesium parvum (E,F) across the different treatments (see Figure 1 for treatment abbreviations). Within each treatment, the reads of each species were compared between the two sequencing methods Illumina MiSeq and Ion Torrent and between filtered and unfiltered marine plankton samples. For the non-prefiltered seawater samples, increasing relative abundances of reads for the target organisms is evident as their concentration is increased in the original sample for every organism except P. australis . A similar pattern is not observed in prefiltered seawater samples. Low levels of cross contamination between the target species ( P. perurans an L. salmonis reads in algal samples) is also observed.

Techniques Used: Sequencing, Concentration Assay

3) Product Images from "Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03009

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Sequencing, Incubation, Isolation

4) Product Images from "A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery"

Article Title: A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery

Journal: bioRxiv

doi: 10.1101/453910

Reverse Transcriptase mediated ribosomal RNA (rRNA) depletion increases target-specific coverage while reducing the number of rRNA reads in next-generation sequencing. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted and DNAse treated. This is considered the Input RNA. DNAse-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNAse I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Duplicate reads were removed using Clumpify and then unique reads were mapped using Bowtie2 to the appropriate reference sequence, 18S rRNA (A), 28S rRNA (B), An. gambiae transcriptome (C), West Nile virus (D) and Bolahun virus (E). Percentage of reads mapping was calculated using MultiQC. Variants detected in Bolahun virus were called using LoFreq (F).
Figure Legend Snippet: Reverse Transcriptase mediated ribosomal RNA (rRNA) depletion increases target-specific coverage while reducing the number of rRNA reads in next-generation sequencing. Anopheles gambiae mosquitoes were exposed to an infectious bloodmeal containing 10 7 PFU of West Nile virus strain NY99. The following day, midguts were dissected and the residual bloodmeal was spread onto a CloneSaver FTA card (GE Healthcare, USA) and then soaked in RNAlater solution to stabilize the nucleic acid and facilitate dispersion. Total nucleic acid was then extracted and DNAse treated. This is considered the Input RNA. DNAse-free RNA was then reverse transcribed using either ribosomal RNA specific probes (RT – with Probes) or without probes (RT – no Probes). The samples were then treated with RNAse H and DNAse I and purified. The samples were then subjected to library preparation and sequenced on an Illumina MiSeq. Reads were then demultiplexed and subsequently trimmed using BBDuk. Duplicate reads were removed using Clumpify and then unique reads were mapped using Bowtie2 to the appropriate reference sequence, 18S rRNA (A), 28S rRNA (B), An. gambiae transcriptome (C), West Nile virus (D) and Bolahun virus (E). Percentage of reads mapping was calculated using MultiQC. Variants detected in Bolahun virus were called using LoFreq (F).

Techniques Used: Next-Generation Sequencing, Purification, Sequencing

5) Product Images from "Cost-effective, high-throughput, single-haplotype iterative mapping and sequencing for complex genomic structures"

Article Title: Cost-effective, high-throughput, single-haplotype iterative mapping and sequencing for complex genomic structures

Journal: Nature protocols

doi: 10.1038/nprot.2018.019

Overview of SHIMS 2.0 protocol. A timeline of a single iteration of the SHIMS 2.0 protocol, showing the major protocol steps, with key quality controls on the right. During a single week-long iteration, 192 clones are processed in parallel, and the resulting draft clone sequences are used to identify sequence family variants (SFVs) that distinguish paralogous ampliconic sequences. A single technician can proceed from a list of clones to completed Illumina libraries in 5 days. After a 2-day long MiSeq run, a bioinformatics specialist assembles demultiplexed fastq sequences into draft clone assemblies and identifies SFVs to select clones for the next iteration.
Figure Legend Snippet: Overview of SHIMS 2.0 protocol. A timeline of a single iteration of the SHIMS 2.0 protocol, showing the major protocol steps, with key quality controls on the right. During a single week-long iteration, 192 clones are processed in parallel, and the resulting draft clone sequences are used to identify sequence family variants (SFVs) that distinguish paralogous ampliconic sequences. A single technician can proceed from a list of clones to completed Illumina libraries in 5 days. After a 2-day long MiSeq run, a bioinformatics specialist assembles demultiplexed fastq sequences into draft clone assemblies and identifies SFVs to select clones for the next iteration.

Techniques Used: Clone Assay, Sequencing

6) Product Images from "Detecting host-parasitoid interactions in an invasive Lepidopteran using nested tagging DNA-metabarcoding"

Article Title: Detecting host-parasitoid interactions in an invasive Lepidopteran using nested tagging DNA-metabarcoding

Journal: bioRxiv

doi: 10.1101/035071

Preparation of PCR amplicon libraries for Illumina MiSeq using a nested metabarcoding approach to detecting species interactions. Colour choices have the same meaning as in Figure 1 . Shades of colours represent the same target sequences in different individuals. Adapted from Evans et al . (2016) .
Figure Legend Snippet: Preparation of PCR amplicon libraries for Illumina MiSeq using a nested metabarcoding approach to detecting species interactions. Colour choices have the same meaning as in Figure 1 . Shades of colours represent the same target sequences in different individuals. Adapted from Evans et al . (2016) .

Techniques Used: Polymerase Chain Reaction, Amplification

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