scholarly journals Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing

2021 ◽  
Vol 5 (2) ◽  
pp. 24
Author(s):  
Dino Pećar ◽  
Ivana Čeko ◽  
Lana Salihefendić ◽  
Rijad Konjhodžić
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Magnetic Beads ◽  
Rna Isolation ◽  
Whole Genome ◽  
Library Preparation ◽  
Rt Pcr ◽  
Sequence Coverage ◽  
Cdna Synthesis ◽  
Good Laboratory

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

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.

scholarly journals Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yury A. Barbitoff ◽  
Dmitrii E. Polev ◽  
Andrey S. Glotov ◽  
Elena A. Serebryakova ◽  
Irina V. Shcherbakova ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Sequence Coverage ◽  
Coding Sequence ◽  
Whole Exome

scholarly journals False COVID-19 cases due to contamination by inactivated virus vaccine

2021 ◽  
Author(s):  
Kelvin Kai-Wang To ◽  
Xin Li ◽  
David Christopher Lung ◽  
Jonathan Daniel Ip ◽  
Wan-Mui Chan ◽  
...  
Keyword(s):  
Public Health ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Virus Vaccine ◽  
False Positive ◽  
Spike Protein ◽  
Whole Genome ◽  
Rt Pcr ◽  
Health Measures ◽  
Respiratory Specimens

Abstract A false-positive SARS-CoV-2 RT-PCR result can lead to unnecessary public-health measures. We report two individuals whose respiratory specimens were contaminated by inactivated SARS-CoV-2 vaccine strain(CoronaVac), likely at vaccination premises. Incidentally, whole-genome sequencing of CoronaVac showed adaptive deletions on the spike protein, which do not result in observable changes of antigenicity.

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

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 Advanced Whole Genome Sequencing Using an Entirely PCR-free Massively Parallel Sequencing Workflow

2019 ◽  
Author(s):  
Hanjie Shen ◽  
Pengjuan Liu ◽  
Zhanqing Li ◽  
Fang Chen ◽  
Hui Jiang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Variant Calling ◽  
Systematic Errors ◽  
Massively Parallel ◽  
Whole Genome ◽  
Library Preparation ◽  
Parallel Sequencing

AbstractBackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling.ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ™ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ™ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ™ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope).ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ™ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

scholarly journals Detection of SARS-CoV-2 from Patient Fecal Samples by Whole Genome Sequencing

2020 ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Analysis ◽  
Nasopharyngeal Swab ◽  
Whole Genome ◽  
Rt Pcr ◽  
Whole Genome Analysis ◽  
Fecal Samples ◽  
Oral Transmission ◽  
Study Participants

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.

scholarly journals Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

2020 ◽  
Vol 92 (11) ◽  
pp. 2725-2734 ◽  
Author(s):  
Wan‐Mui Chan ◽  
Jonathan Daniel Ip ◽  
Allen Wing‐Ho Chu ◽  
Cyril Chik‐Yan Yip ◽  
Lap‐Sum Lo ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Rt Pcr ◽  
Nsp1 Gene

scholarly journals Emergence of SARS-CoV-2 variant B.1.575.2 containing the E484K mutation in the spike protein in Pamplona (Spain) May-June 2021

2021 ◽  
Author(s):  
Camino Trobajo-Sanmartín ◽  
Ana Miqueleiz ◽  
María Eugenia Portillo ◽  
Miguel Fernández-Huerta ◽  
Ana Navascués ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Threshold Value ◽  
Spike Protein ◽  
Whole Genome ◽  
Viral Dynamics ◽  
Rt Pcr ◽  
Northern Spain ◽  
Novel Mutations ◽  
Rapid Control

With the emergence of new SARS-CoV-2 variants and the acquisition of novel mutations in exiting lineages, the need to implement methods capable of monitoring viral dynamics arises. We report the emergence and spread of a new SARS-CoV-2 variant within B.1.575 lineage containing the E484K mutation in the spike protein (named B.1.575.2) in a region of Northern Spain between May and June 2021. SARS-CoV-2 positive samples with cycle threshold value less than or equal to 30 were selected to screen of presumptive variants using the TaqPathTM COVID-19 RT-PCR kit and TaqManTM SARS-CoV-2 Mutation Panel. Confirmation of variants was performed by whole genome sequencing. Of the 200 samples belonging to the B.1.575 lineage, 194 (97%) corresponded to the B.1.575.2 sub-lineage, which was related to the presence of the E484K mutation. Of 197 cases registered in GISAID EpiCoV database as lineage B.1.575.2 194 (99.5%) were identified in Pamplona (Spain). This report emphasizes the importance of complementing surveillance of SARS-CoV-2 with sequencing for the rapid control of emerging viral variants.

scholarly journals Museomics of a rare taxon: placing Whalleyanidae in the Lepidoptera Tree of Life

2020 ◽  
Author(s):  
Victoria G. Twort ◽  
Joël Minet ◽  
Christopher W. Wheat ◽  
Niklas Wahlberg
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
De Novo ◽  
Sister Group ◽  
Sister Group Relationship ◽  
Whole Genome ◽  
Museum Specimens ◽  
Library Preparation ◽  
Natural History Museums ◽  
History Museums

AbstractMuseomics is a valuable tool that utilises the diverse biobanks that are natural history museums. The ability to sequence genomes from old specimens has expanded not only the variety of interesting taxa available to study but also the scope of questions that can be investigated in order to further knowledge about biodiversity. Here we present whole genome sequencing results from the enigmatic genus Whalleyana, as well as the families Callidulidae and Hyblaeidae. Library preparation was carried out on four museum specimens and one existing DNA extract and sequenced with Illumina short reads. De novo assembly resulted in highly fragmented genomes with the N50 ranging from 317 – 2,078 bp. Mining of a manually curated gene set of 332 genes from these draft genomes had an overall gene recovery rate of 64 – 90%. Phylogenetic analysis places Whalleyana as sister to Callidulidae, and Hyblaea as sister to Pyraloidea. Since the former sister-group relationship turns out to be also supported by ten morphological synapomorphies, we propose to formally assign the Whalleyanidae to the superfamily Calliduloidea. These results highlight the usefulness of not only museum specimens, but also existing DNA extracts, for whole genome sequencing and gene mining for phylogenomic studies.

Sign in / Sign up

Export Citation Format

Share Document