Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The proposed protocol allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titer virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.

A high scale SARS-CoV-2 profiling by its whole-genome sequencing using Oxford Nanopore Technology in Kazakhstan.

Severe acute respiratory syndrome (SARS-CoV-2) is responsible for the worldwide pandemic, COVID-19. The original viral whole-genome was sequenced by a high-throughput sequencing approach from the samples obtained from Wuhan, China. Real-time gene sequencing is the main parameter to manage viral outbreaks because it expands our understanding of virus proliferation, spread, and evolution. Whole-genome sequencing is critical for SARS-CoV-2 variant surveillance, the development of new vaccines and boosters, and the representation of epidemiological situations in the country. A significant increase in the number of COVID-19 cases confirmed in August 2021 in Kazakhstan facilitated a need to establish an effective and proficient system for further study of SARS-CoV-2 genetic variants and the development of future Kazakhstan's genomic surveillance program. The SARS-CoV-2 whole-genome was sequenced according to SARS-CoV-2 ARTIC protocol (EXP-MRT001) by Oxford Nanopore Technologies at the National Laboratory Astana, Kazakhstan to track viral variants circulating in the country. The 500 samples kindly provided by the Republican Diagnostic Center (UMC-NU) and private laboratory KDL "Olymp" were collected from individuals in Nur-Sultan city diagnosed with COVID-19 from August 2021 to May 2022 using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). All samples had a cycle threshold (Ct) value below 20 with an average Ct value of 17.03. The overall average value of sequencing depth coverage for samples is 244X. 341 whole-genome sequences that passed quality control were deposited in the Global initiative on sharing all influenza data (GISAID). The BA.1.1 (n = 189), BA.1 (n = 15), BA.2 (n = 3), BA.1.15 (n = 1), BA.1.17.2 (n = 1) omicron lineages, AY.122 (n = 119), B.1.617.2 (n = 8), AY.111 (n = 2), AY.126 (n = 1), AY.4 (n = 1) delta lineages, one sample B.1.1.7 (n = 1) belongs to alpha lineage, and one sample B.1.637 (n = 1) belongs to small sublineage were detected in this study. This is the first study of SARS-CoV-2 whole-genome sequencing by the ONT approach in Kazakhstan, which can be expanded for the investigation of other emerging viral or bacterial infections on the country level.

re-Searcher: GUI-based bioinformatics tool for simplified genomics data mining of VCF files.

BACKGROUND: High-throughput sequencing platforms generate a massive amount of high-dimensional genomic datasets that are available for analysis. Modern and user-friendly bioinformatics tools for analysis and interpretation of genomics data becomes essential during the analysis of sequencing data. Different standard data types and file formats have been developed to store and analyze sequence and genomics data. Variant Call Format (VCF) is the most widespread genomics file type and standard format containing genomic information and variants of sequenced samples. RESULTS: Existing tools for processing VCF files don't usually have an intuitive graphical interface, but instead have just a command-line interface that may be challenging to use for the broader biomedical community interested in genomics data analysis. re-Searcher solves this problem by pre-processing VCF files by chunks to not load RAM of computer. The tool can be used as standalone user-friendly multiplatform GUI application as well as web application (https://nla-lbsb.nu.edu.kz). The software including source code as well as tested VCF files and additional information are publicly available on the GitHub repository (https://github.com/LabBandSB/re-Searcher).

Whole-genome sequencing data of Kazakh individuals.

OBJECTIVES: Kazakhstan is a Central Asian crossroad of European and Asian populations situated along the way of the Great Silk Way. The territory of Kazakhstan has historically been inhabited by nomadic tribes and today is the multi-ethnic country with the dominant Kazakh ethnic group. We sequenced and analyzed the whole-genomes of five ethnic healthy Kazakh individuals with high coverage using next-generation sequencing platform. This whole-genome sequence data of healthy Kazakh individuals can be a valuable reference for biomedical studies investigating disease associations and population-wide genomic studies of ethnically diverse Central Asian region. DATA DESCRIPTION: Blood samples have been collected from five ethnic healthy Kazakh individuals living in Kazakhstan. The genomic DNA was extracted from blood and sequenced. Sequencing was performed on Illumina HiSeq2000 next-generation sequencing platform. We sequenced and analyzed the whole-genomes of ethnic Kazakh individuals with the coverage ranging from 26 to 32X. Ranging from 98.85 to 99.58% base pairs were totally mapped and aligned on the human reference genome GRCh37 hg19. Het/Hom and Ts/Tv ratios for each whole genome ranged from 1.35 to 1.49 and from 2.07 to 2.08, respectively. Sequencing data are available in the National Center for Biotechnology Information SRA database under the accession number PRJNA374772.