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

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.

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.

scholarly journals Whole Genome Sequencing and Antimicrobial Resistance of Staphylococcus aureus from Surgical Site Infections in Ghana

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 196
Author(s):  
Beverly Egyir ◽  
Jeannette Bentum ◽  
Naiki Attram ◽  
Anne Fox ◽  
Noah Obeng-Nkrumah ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Meca Gene ◽  
Surgical Site Infections ◽  
Illumina Miseq ◽  
Multi Drug Resistance ◽  
Toxin Gene ◽  
Whole Genome ◽  
Flight Mass Spectrometry

Staphylococcus aureus (S. aureus) is a common cause of surgical site infections (SSIs) globally. Data on the occurrence of methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) among patients with surgical site infections (SSIs) in sub-Saharan African are scarce. We characterized S. aureus from SSIs in Ghana using molecular methods and antimicrobial susceptibility testing (AST). Wound swabs or aspirate samples were collected from subjects with SSIs. S. aureus was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS); AST was performed by Kirby-Bauer disk diffusion, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Detection of spa, mecA, and pvl genes was performed by polymerase chain reaction (PCR). Whole-genome sequencing (WGS) was done using the Illumina MiSeq platform. Samples were collected from 112 subjects, with 13 S. aureus isolates recovered. Of these, 92% were sensitive to co-trimoxazole, 77% to clindamycin, and 54% to erythromycin. Multi-drug resistance was detected in 5 (38%) isolates. The four mecA gene-positive MRSA isolates detected belonged to ST152 (n = 3) and ST5 (n = 1). In total, 62% of the isolates were positive for the Panton-Valentine leukocidin (pvl) toxin gene. This study reports, for the first time, a pvl-positive ST152-t355 MRSA clone from SSIs in Ghana. The occurrence of multi-drug-resistant S. aureus epidemic clones suggests that continuous surveillance is required to monitor the spread and resistance trends of S. aureus in hospital settings in the country.

Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v2

2022 ◽  
Author(s):  
jason.nguyen not provided ◽  
Tracy Lee ◽  
Rebecca Hickman ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Sequencing Platforms

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw

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.

Whole-Genome Sequencing: Automated, Nonindexed Library Preparation

2016 ◽  
Vol 2017 (3) ◽  
pp. pdb.prot094623 ◽  
Author(s):  
Elaine Mardis ◽  
W. Richard McCombie
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Library Preparation

scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2020 ◽  
Vol 280 ◽  
pp. 113865 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Ion Torrent ◽  
Ion Torrent Pgm

scholarly journals Whole-Genome Sequencing of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador

2020 ◽  
Vol 9 (13) ◽  
Author(s):  
William Calero-Cáceres ◽  
Joyce Villacís ◽  
Maria Ishida ◽  
Elton Burnett ◽  
Christian Vinueza-Burgos
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Egg Production ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Poultry Farms

Five strains of Salmonella enterica subsp. enterica serovar Infantis and two strains of S. enterica subsp. enterica serovar Kentucky isolated in 2017 from Ecuadorian layer poultry farms were sequenced using Illumina MiSeq technology. These isolates were collected on layer farms in central Ecuador, one of the most important areas of egg production in the country. The genome sequences of these isolates show valuable information for surveillance purposes.

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