Sequence analysis viewer, by Illumina - Product details

1

Whole-Genome Sequencing and Somatic Variant Identification

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Genomic DNA was isolated from 5 × 10⁶ cells using the DNA Purification Kit (Promega). Whole-genome sequencing (WGS) was performed and analyzed at the Yale Center for Genome Analysis (West Haven, CT). Using Illumina's Sequence Analysis Viewer, various quality control (QC) parameters were monitored to assess the sample quality. The sequencing reads passing the QC parameter values were aligned to the hs38DH human reference for variant calling. The GATK MuTect2 was used to call and filter somatic variants.

Jung M., Kowalczyk K., Hankins R., Bandi G., Kallakury B., Carrasquilla M.A., Banerjee P.P., Grindrod S, & Dritschilo A. (2022). Novel Paired Normal Prostate and Prostate Cancer Model Cell Systems Derived from African American Patients. Cancer Research Communications, 2(12), 1617-1625.

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2

Phage Library Immunoprecipitation and Sequencing

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The T7-HCoV-56-mer phage library was generously provided to us by Dr. Stephen J. Elledge at Harvard Medical School, and it was previously described [29 (link)]. Phage immunoprecipitation and sequencing (PhIP-Seq) have been previously described by us and others [26 (link),27 (link),29 (link),50 (link)]. Briefly, the library was amplified and titered per the manufacturer’s instructions (Novagen T7Select System, Burlington, MA, USA). Then, 1.4 × 10⁹ pfu of the library (≈2 × 10⁵ pfu/library member) was mixed with plasma containing 2 µg of total IgG. The phage–antibody complexes were immunoprecipitated using Protein A and Protein G magnetic beads (10008D/9D, Invitrogen, Boston, MA, USA) and a magnetic separation rack (S1511S, NEB, Ipswich, MA, USA). After immunoprecipitation, the beads were resuspended in 40 µL of nuclease-free water, heated to 95 °C to lyse the phage, and the phage DNA was PCR amplified. During PCR amplification, each well of the 96-well plate was barcoded, and Illumina adaptors were added. The 376bp amplicon was then gel-extracted and sent to the UNMC Genomics core for quality check and sequencing. Finally, the clustering and sequencing were performed on an Illumina NextSeq550 using the 50-cycle, single-end protocol (Mid-output flow-cell). The run was monitored by Illumina Sequence Analysis Viewer, and the final FASTQ files were generated after de-multiplexing.

Yalcin D., Bennett S.J., Sheehan J., Trauth A.J., Tso F.Y., West J.T., Hagensee M.E., Ramsay A.J, & Wood C. (2023). Longitudinal Variations in Antibody Responses against SARS-CoV-2 Spike Epitopes upon Serial Vaccinations. International Journal of Molecular Sciences, 24(8), 7292.

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3

Small RNA Sequencing Library Preparation

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Cluster generation of the small RNA sequencing libraries and a PhiX control library was performed using an automated cBot system (Illumina). All sequencing libraries were loaded onto a TruSeq single-read flow cell v3 at a concentration of 12 pM. The small RNA sequencing libraries were spiked with 1% (vol/vol) PhiX control library and loaded onto four individual flow cell lanes based on the tissue of origin as each lane contained multiplexed sequencing libraries. The small RNA libraries were sequenced using the Illumina HiScan SQ system for a total of 58 cycles. The performance of the sequencing run and quality of raw sequence data were assessed based on the reports created by the Illumina Sequence Analysis Viewer. All reported parameters met the quality assessment criteria recommended by the manufacturer.

Permenter M.G., McDyre B.C., Ippolito D.L, & Stallings J.D. (2019). Alterations in tissue microRNA after heat stress in the conscious rat: potential biomarkers of organ-specific injury. BMC Genomics, 20, 141.

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4

Whole Genome Sequencing Using Illumina Platforms

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WGS was performed on either the Illumina HiSeq2000 or the HighSeq2500 run in standard mode, either by the Oxford Genomics Centre at the Wellcome Trust Centre for Human Genetics, or by Illumina Cambridge Ltd.. We generated 100 bp reads and used v2.5 or v3 clustering and sequencing chemistry. A PhiX control was spiked into the libraries. We aimed for a mean coverage of 30× and obtained a minimum of 22.7×. The mode number of lanes required to reach the desired coverage was 2⅓.
We used the recommended quality metrics in the Illumina Sequence Analysis Viewer in analysing each lane. Additionally, we generated our own quality metrics for each lane (or, in the case of multiplexes, each part of a lane), and required the following criteria to be met: <2% duplicate pairs; most frequent kmer <2%; >99% mapped; <2.5% read pairs mapping to different chromosomes; mean insert size between 340 bp and 440 bp, with a median absolute deviation of <30bp; approximately uniform genomic coverage by GC content; ~1% exonic coverage; <2% N bases at any cycle; approximately equal number of reads per tag (three samples multiplexed per lane), standard deviation <25%.

Taylor J.C., Martin H.C., Lise S., Broxholme J., Cazier J.B., Rimmer A., Kanapin A., Lunter G., Fiddy S., Allan C., Aricescu A.R., Attar M., Babbs C., Becq J., Beeson D., Bento C., Bignell P., Blair E., Buckle V.J., Bull K., Cais O., Cario H., Chapel H., Copley R.R., Cornall R., Craft J., Dahan K., Davenport E.E., Dendrou C., Devuyst O., Fenwick A.L., Flint J., Fugger L., Gilbert R.D., Goriely A., Green A., Greger I.H., Grocock R., Gruszczyk A.V., Hastings R., Hatton E., Higgs D., Hill A., Holmes C., Howard M., Hughes L., Humburg P., Johnson D., Karpe F., Kingsbury Z., Kini U., Knight J.C., Krohn J., Lamble S., Langman C., Lonie L., Luck J., McCarthy D., McGowan S.J., McMullin M.F., Miller K.A., Murray L., Németh A.H., Nesbit M.A., Nutt D., Ormondroyd E., Oturai A.B., Pagnamenta A., Patel S.Y., Percy M., Petousi N., Piazza P., Piret S.E., Polanco-Echeverry G., Popitsch N., Powrie F., Pugh C., Quek L., Robbins P.A., Robson K., Russo A., Sahgal N., van Schouwenburg P.A., Schuh A., Silverman E., Simmons A., Sørensen P.S., Sweeney E., Taylor J., Thakker R.V., Tomlinson I., Trebes A., Twigg S.R., Uhlig H.H., Vyas P., Vyse T., Wall S.A., Watkins H., Whyte M.P., Witty L., Wright B., Yau C., Buck D., Humphray S., Ratcliffe P.J., Bell J.I., Wilkie A.O., Bentley D., Donnelly P, & McVean G. (2015). Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nature genetics, 47(7), 717-726.

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5

Whole Genome Sequencing Using Illumina Platforms

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WGS was performed on either the Illumina HiSeq2000 or the HighSeq2500 run in standard mode, either by the Oxford Genomics Centre at the Wellcome Trust Centre for Human Genetics, or by Illumina Cambridge Ltd.. We generated 100 bp reads and used v2.5 or v3 clustering and sequencing chemistry. A PhiX control was spiked into the libraries. We aimed for a mean coverage of 30× and obtained a minimum of 22.7×. The mode number of lanes required to reach the desired coverage was 2⅓.
We used the recommended quality metrics in the Illumina Sequence Analysis Viewer in analysing each lane. Additionally, we generated our own quality metrics for each lane (or, in the case of multiplexes, each part of a lane), and required the following criteria to be met: <2% duplicate pairs; most frequent kmer <2%; >99% mapped; <2.5% read pairs mapping to different chromosomes; mean insert size between 340 bp and 440 bp, with a median absolute deviation of <30bp; approximately uniform genomic coverage by GC content; ~1% exonic coverage; <2% N bases at any cycle; approximately equal number of reads per tag (three samples multiplexed per lane), standard deviation <25%.

Taylor J.C., Martin H.C., Lise S., Broxholme J., Cazier J.B., Rimmer A., Kanapin A., Lunter G., Fiddy S., Allan C., Aricescu A.R., Attar M., Babbs C., Becq J., Beeson D., Bento C., Bignell P., Blair E., Buckle V.J., Bull K., Cais O., Cario H., Chapel H., Copley R.R., Cornall R., Craft J., Dahan K., Davenport E.E., Dendrou C., Devuyst O., Fenwick A.L., Flint J., Fugger L., Gilbert R.D., Goriely A., Green A., Greger I.H., Grocock R., Gruszczyk A.V., Hastings R., Hatton E., Higgs D., Hill A., Holmes C., Howard M., Hughes L., Humburg P., Johnson D., Karpe F., Kingsbury Z., Kini U., Knight J.C., Krohn J., Lamble S., Langman C., Lonie L., Luck J., McCarthy D., McGowan S.J., McMullin M.F., Miller K.A., Murray L., Németh A.H., Nesbit M.A., Nutt D., Ormondroyd E., Oturai A.B., Pagnamenta A., Patel S.Y., Percy M., Petousi N., Piazza P., Piret S.E., Polanco-Echeverry G., Popitsch N., Powrie F., Pugh C., Quek L., Robbins P.A., Robson K., Russo A., Sahgal N., van Schouwenburg P.A., Schuh A., Silverman E., Simmons A., Sørensen P.S., Sweeney E., Taylor J., Thakker R.V., Tomlinson I., Trebes A., Twigg S.R., Uhlig H.H., Vyas P., Vyse T., Wall S.A., Watkins H., Whyte M.P., Witty L., Wright B., Yau C., Buck D., Humphray S., Ratcliffe P.J., Bell J.I., Wilkie A.O., Bentley D., Donnelly P, & McVean G. (2015). Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nature genetics, 47(7), 717-726.

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6

Illumina MiSeq Sequencing Data Analysis

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Imaging, data processing, and evaluation of the sequencing run performance were carried out using the Illumina MiSeq Reporter (MSR; v2.5.1.3) and the Illumina Sequence Analysis Viewer (SAV; v 2.4.7). The initial QC and 3′-end adapter trimming were performed using the MiSeq inherited MSR software packages. This was followed by demultiplexing of passed filter reads based on their indices and corresponding sample IDs. The resulting R1 and R2 (.fastq) files, containing quality values and sequence information, were used for downstream analyses.

Gupta A, & Nair S. (2022). Heritable Epigenomic Modifications Influence Stress Resilience and Rapid Adaptations in the Brown Planthopper (Nilaparvata lugens). International Journal of Molecular Sciences, 23(15), 8728.

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7

16S rRNA Amplicon Sequencing Protocol

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PCR products were purified and recovered by a DNA Gel Recovery Kit in accordance with the previously validated protocol. The DNA samples were amplified with a 16S Metagenomic Sequencing Library Preparation Kit (Illumina, CA, USA). 16S rRNA V4 region was amplified in two step PCR protocols. PCR amplicon was performed with a gel extraction kit (Nippon Genetics, Japan). The sequences of the primer used were as follows: F: 5'-GTGCCAGCMG-CCGCGG-3'; R: 5'-GGACTACHVGGGTWTCTAAT-3'. The library validation was conducted using the Illumina MiSeq system. Sequencing was conducted with HiSeq 2500 PE250 and analyzed with Sequence Analysis Viewer (Illumina).

Liang C., Xu D., Zhang M., Zhang P., Guan Y., Kzhyshkowska J., & Liang C. (2021). Urine flora imbalance and new biomarkers in prostate cancer and benign prostatic hyperplasia. Archives of Medical Science.

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Sequence analysis viewer

Lab products found in correlation

7 protocols using sequence analysis viewer

Whole-Genome Sequencing and Somatic Variant Identification

Phage Library Immunoprecipitation and Sequencing

Small RNA Sequencing Library Preparation

Whole Genome Sequencing Using Illumina Platforms

Whole Genome Sequencing Using Illumina Platforms

Illumina MiSeq Sequencing Data Analysis

16S rRNA Amplicon Sequencing Protocol

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