SeqPlus sequencing methodology enables robust whole-genome sequencing, true variant detection, and novel genomic insights from archival esophageal carcinoma FFPE samples.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13016-e13016
Author(s):  
Shannon Terrell Bailey ◽  
Belynda Hicks ◽  
Bin Zhu ◽  
Nan Hu ◽  
Phil R. Taylor ◽  
...  
Keyword(s):  
Esophageal Carcinoma ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Limit Of Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
High Quality ◽  
Primary Tumors ◽  
Ffpe Samples ◽  
Fresh Frozen

e13016 Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage & sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction & library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors & matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) & identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage & average duplicate reads were 31%. We noted similar transition/transversion ratios & mutational spectra as from fresh-frozen EC specimens, suggesting that extraction & library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs & indels derived from WGS & targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] > 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels & CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical & outcomes data.

Abstract 427: High-quality whole-genome sequencing of FFPE samples

2018 ◽  
Author(s):  
Shannon T. Bailey ◽  
Jim Lund ◽  
Hao Wang ◽  
Weiren Cui ◽  
Chen Hao ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types

2019 ◽  
Author(s):  
Shannon T. Bailey ◽  
James Lund ◽  
Muhammad Ekram ◽  
Wanfeng Yu ◽  
Richard T. Williams ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types

2019 ◽  
Author(s):  
Shannon T. Bailey ◽  
James Lund ◽  
Muhammad Ekram ◽  
Wanfeng Yu ◽  
Richard T. Williams ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

High-quality whole-genome sequencing of FFPE samples.

2018 ◽  
Vol 36 (15_suppl) ◽  
pp. e13500-e13500 ◽  
Author(s):  
Shannon Terrell Bailey ◽  
Jim Lund ◽  
Hao Wang ◽  
Hao Chen ◽  
Hongye Sun ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

Low-coverage whole genome sequencing of FFPE-derived-DNA and extracellular vesicle-associated DNA in patients with metastatic cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14523-e14523
Author(s):  
Bella Hai Nguyen ◽  
Nick Wong ◽  
Timothy Semple ◽  
Olivia Ruhen ◽  
Stephen Q Wong ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Metastatic Cancer ◽  
Valuable Insight ◽  
Whole Genome ◽  
Plasma Dna ◽  
Primary Tumors ◽  
Hpv Status ◽  
Ffpe Samples ◽  
Low Coverage

e14523 Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log2 ratio ≥0.15 and losses < -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.

Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer v2

2021 ◽  
Author(s):  
Julie Haendiges ◽  
Narjol Gonzalez-Escalona ◽  
Ruth E Timme ◽  
Maria Balkey
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Library Preparation ◽  
High Quality ◽  
Dna Quantitation ◽  
Quality Checks ◽  
Automated Dna Extraction

This procedure outlines the protocol for whole genome sequencing of bacterial organisms using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network. Complete in order: 1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube) Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing 2. DNA Quantitation Quantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Included SOP or Library Preparation using Illumina Nextera XT ) Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex) 4. Sequencing using Illumina MiSeq 5. Data Quality Checks and NCBI Submission

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

2022 ◽  
Author(s):  
Jason Nguyen ◽  
Rebecca Hickman ◽  
Tracy Lee ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Library Preparation ◽  
Dna Libraries ◽  
Reaction Volumes ◽  
Single Size ◽  
Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

scholarly journals Whole-Genome Sequencing Applied to the Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli O157:H7 in Argentina: TABLE 1 

2016 ◽  
Vol 4 (6) ◽  
Author(s):  
Claudia Carolina Carbonari ◽  
Nahuel Fittipaldi ◽  
Sarah Teatero ◽  
Taryn B. T. Athey ◽  
Luis Pianciola ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Shiga Toxin ◽  
Whole Genome ◽  
Escherichia Coli O157 ◽  
Genome Sequences ◽  
High Quality ◽  
Human Infections

Shiga toxin-producing Escherichia coli strains are worldwide associated with sporadic human infections and outbreaks. In this work, we report the availability of high-quality draft whole-genome sequences for 19 O157:H7 strains isolated in Argentina.

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