scholarly journals SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing

2020 ◽  
Author(s):  
AS Speranskaya ◽  
VV Kaptelova ◽  
AV Valdokhina ◽  
VP Bulanenko ◽  
AE Samoilov ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Preparation Method ◽  
Virus Genome ◽  
Clinical Samples ◽  
Full Genome ◽  
Library Preparation ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Technical Data ◽  
Library Preparation Method

ABSTRACTHere we provide technical data for amplifying the complete genome of SARS-CoV-2 from clinical samples using only seventeen pairs of primers. We demonstrate that the СV2000bp primer panel successfully produces genomes when used with the residual total RNA extracts from positive clinical samples following diagnostic RT-PCRs (with Ct in the range from 13 to 20). The library preparation method reported here includes genome amplification of ~1750-2000 bp fragments followed by ultrasonic fragmentation combined with the introduction of Illumina compatible adapters. Using the SCV2000bp panel, 25 complete SARS-CoV-2 virus genome sequences were sequenced from clinical samples of COVID-19 patients from Moscow obtained in late March - early April.

scholarly journals Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Steven Van Borm ◽  
Toon Rosseel ◽  
Andy Haegeman ◽  
Mpolokang Elliot Fana ◽  
Latoa Seoke ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Complete Genome ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Clinical Samples ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Foot And Mouth

The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals.

scholarly journals Two-Pool Multiplex Long-Amplicon Amplification and Full-Genome Sequencing of SARS-CoV-2

2020 ◽  
Author(s):  
Hong-jie Liu ◽  
Jin-Hui Li ◽  
Yan-Feng Lin ◽  
Xiao-Chen Bo ◽  
Hong-Bin Song ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Genomic Variations ◽  
Pcr Targeting

Abstract BackgroundSince the first public genome of SARS-CoV-2, over 170,000 genome sequences of the virus have been shared by researchers world-wide (till November 1st 2020). Multiplex PCR targeting SARS-CoV-2 followed by massively parallel sequencing (MPS) and/or nanopore sequencing is a widely used strategy to recover the genome from primary samples. However, the bias of amplification among different amplicons should not be ignored, which might lead to uneven sequencing coverage on the viral genome.MethodsWe aim to develop a novel multiplex PCR panel to achieve an improved coverage evenness of SARS-CoV-2. We adapt long amplicons (~1000-bp) for the panel and thus reduced the number of primer pairs. The panel was validated with clinical samples and sequenced via MPS sequencing systems and a portable nanopore sequencing device MinION. We evaluated the full-genome coverage evenness and its dependence on viral loads of the long amplicon panel; we then compared it with a 98-plex panel provided by the ARTIC network. The accuracy to identify viral genomic variations based on the panel and sequencing with MinION was assessed.ResultsWe developed a two-pool 36-plex panel for full-genome sequencing of SARS-CoV-2, whose amplicon size ranged from 880 to 1027 bp. For samples with a <30 Ct value, >90% viral genome could be recovered with a high sequencing depth (>0.2 mean depth) by using the long-amplicon panel (n = 36), compared with 79-88% highly covered genome region for the ARTIC panel (n = 5). The coverage evenness of the long-amplicon panel was also less affected by low viral titers and not dependent on sequencing data amount. With MinION sequencing, the consensus viral genomes could be reliably recovered. However, a high false positive rate was observed to identify sub-clonal genomic variations with a <0.6 frequency.ConclusionA novel multiplex PCR panel for full-genome sequencing of SARS-CoV-2 with improved coverage evenness and low requirement of data throughput was validated with clinical samples. Amplification of SARS-CoV-2 with the panel followed by MinION sequencing could generate reliable consensus genome sequences, but the detection of non-dominating viral populations within host is error-prone.

scholarly journals A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Emily A Saunderson ◽  
Ann-Marie Baker ◽  
Marc Williams ◽  
Kit Curtius ◽  
J Louise Jones ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Preparation Method ◽  
Degraded Dna ◽  
Whole Genome ◽  
Library Preparation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Library Preparation Method

Abstract The desire to analyse limited amounts of biological material, historic samples and rare cell populations has collectively driven the need for efficient methods for whole genome sequencing (WGS) of limited amounts of poor quality DNA. Most protocols are designed to recover double-stranded DNA (dsDNA) by ligating sequencing adaptors to dsDNA with or without subsequent polymerase chain reaction amplification of the library. While this is sufficient for many applications, limited DNA requires a method that can recover both single-stranded DNA (ssDNA) and dsDNA. Here, we present a WGS library preparation method, called ‘degraded DNA adaptor tagging’ (DDAT), adapted from a protocol designed for whole genome bisulfite sequencing. This method uses two rounds of random primer extension to recover both ssDNA and dsDNA. We show that by using DDAT we can generate WGS data from formalin-fixed paraffin-embedded (FFPE) samples using as little as 2 ng of highly degraded DNA input. Furthermore, DDAT WGS data quality was higher for all FFPE samples tested compared to data produced using a standard WGS library preparation method. Therefore, the DDAT method has potential to unlock WGS data from DNA previously considered impossible to sequence, broadening opportunities to understand the role of genetics in health and disease.

scholarly journals Full-Genome Sequencing of a Virus from a 33-Year-Old Sample Demonstrates that Arracacha Mottle Virus Is Synonymous with Arracacha Virus Y

2018 ◽  
Vol 7 (21) ◽  
Author(s):  
Ian P. Adams ◽  
Neil Boonham ◽  
Roger A. C. Jones
Keyword(s):  
Genome Sequencing ◽  
Genome Sequence ◽  
Mottle Virus ◽  
Nucleotide Identity ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Content Type

We describe here the first genome sequence of Arracacha virus Y (ArVY) derived from an arracacha (Arracacia xanthorrhiza) sample originally collected in 1976 in Peru and compare it with other potyvirus genome sequences. It had a 79% nucleotide identity with a 2013 Brazilian Arracacha mottle virus (AMoV) sequence, suggesting that AMoV is ArVY.

scholarly journals Multiplex Sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

2021 ◽  
Author(s):  
Kim-Kee Tan ◽  
VunJia Tiong ◽  
Tan Jia-Yi ◽  
Jo-Ern Wong ◽  
Boon-Teong Teoh ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Clinical Samples ◽  
Personal Genome ◽  
Full Genome ◽  
Personal Genome Machine ◽  
Full Genome Sequencing ◽  
Sequence Coverage ◽  
Genome Variations ◽  
Ion Pgm ◽  
Multiplex Sequencing

Abstract Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13751-13965 and 23941-24106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64-Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13730 and 23929, whereas the other isolates possess T at these positions. The genetic variations observed suggest that the naturally occurring genome variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel. The possible impact of other genetic sequence variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel, hence, should be considered.

scholarly journals SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

2016 ◽  
Vol 45 (6) ◽  
pp. e36-e36 ◽  
Author(s):  
Amanda Raine ◽  
Erika Manlig ◽  
Per Wahlberg ◽  
Ann-Christine Syvänen ◽  
Jessica Nordlund
Keyword(s):  
Preparation Method ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulphite Sequencing ◽  
Library Preparation Method

474 Hepatitis B virus genomic markers for hepatocellular carcinoma on full-genome sequencing of 332 patients

2006 ◽  
Vol 44 ◽  
pp. S176-S177
Author(s):  
J.J.Y. Sung ◽  
S.K.W. Tsui ◽  
C.H. Tse ◽  
E.Y.T. Ng ◽  
K.S. Leung ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Sequencing ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
B Virus ◽  
Genomic Markers

Transcriptome-wide m6A detection with DART-Seq

2019 ◽  
Author(s):  
Kate D. Meyer
Keyword(s):  
Gene Expression Regulation ◽  
Preparation Method ◽  
Current Knowledge ◽  
Cross Reactivity ◽  
High Variability ◽  
Library Preparation ◽  
Rna Seq ◽  
Generation Sequencing ◽  
Library Preparation Method

Abstract m6A is the most abundant internal mRNA modification and plays diverse roles in gene expression regulation. Much of our current knowledge about m6A has been driven by recent advances in the ability to detect this mark transcriptome-wide. Antibody-based approaches have been the method of choice for global m6A mapping studies. These methods rely on m6A antibodies to immunoprecipitate methylated RNAs, followed by next-generation sequencing to identify m6A-containing transcripts1,2. While these methods enabled the first identification of m6A sites transcriptome-wide and have dramatically improved our ability to study m6A, they suffer from several limitations. These include requirements for high amounts of input RNA, costly and time-consuming library preparation, high variability across studies, and m6A antibody cross-reactivity with other modifications. Here, we describe DART-Seq (deamination adjacent to RNA modification targets), an antibody-free method for global m6A detection. In DART-Seq, the C to U deaminating enzyme, APOBEC1, is fused to the m6A-binding YTH domain. This fusion protein is then introduced to cellular RNA either through overexpression in cells or with in vitro assays, and subsequent deamination of m6A-adjacent cytidines is then detected by RNA sequencing to identify m6A sites. DART-Seq can successfully map m6A sites throughout the transcriptome using as little as 10 nanograms of total cellular RNA, and it is compatible with any standard RNA-seq library preparation method.

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