Combination of Sanger and target-enrichment markers supports revised generic delimitation in the problematic ‘Urera clade’ of the nettle family (Urticaceae)

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

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SAUTE: sequence assembly using target enrichment

BMC Bioinformatics ◽

10.1186/s12859-021-04174-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Alexandre Souvorov ◽

Richa Agarwala

Keyword(s):

De Bruijn Graph ◽

Challenging Problem ◽

Target Enrichment ◽

Rna Seq ◽

Sequencing Technology ◽

Insert Size ◽

Systematic Biases ◽

Target Sequences ◽

Genomic Regions ◽

Higher Sensitivity

Abstract Background Illumina is the dominant sequencing technology at this time. Short length, short insert size, some systematic biases, and low-level carryover contamination in Illumina reads continue to make assembly of repeated regions a challenging problem. Some applications also require finding multiple well supported variants for assembled regions. Results To facilitate assembly of repeat regions and to report multiple well supported variants when a user can provide target sequences to assist the assembly, we propose SAUTE and SAUTE_PROT assemblers. Both assemblers use de Bruijn graph on reads. Targets can be transcripts or proteins for RNA-seq reads and transcripts, proteins, or genomic regions for genomic reads. Target sequences are nucleotide and protein sequences for SAUTE and SAUTE_PROT, respectively. Conclusions For RNA-seq, comparisons with Trinity, rnaSPAdes, SPAligner, and SPAdes assembly of reads aligned to target proteins by DIAMOND show that SAUTE_PROT finds more coding sequences that translate to benchmark proteins. Using AMRFinderPlus calls, we find SAUTE has higher sensitivity and precision than SPAdes, plasmidSPAdes, SPAligner, and SPAdes assembly of reads aligned to target regions by HISAT2. It also has better sensitivity than SKESA but worse precision.

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The best of both worlds: Combining lineage‐specific and universal bait sets in target‐enrichment hybridization reactions

Applications in Plant Sciences ◽

10.1002/aps3.11438 ◽

2021 ◽

Author(s):

Kasper P. Hendriks ◽

Terezie Mandáková ◽

Nikolai M. Hay ◽

Elfy Ly ◽

Alex Hooft van Huysduynen ◽

...

Keyword(s):

Target Enrichment

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A target enrichment probe set for resolving the flagellate land plant tree of life

Applications in Plant Sciences ◽

10.1002/aps3.11406 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Jesse W. Breinholt ◽

Sarah B. Carey ◽

George P. Tiley ◽

E. Christine Davis ◽

Lorena Endara ◽

...

Keyword(s):

Land Plant ◽

Tree Of Life ◽

Target Enrichment ◽

Probe Set

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Taxonomic notes on the genus Hedyotis sensu lato (Rubiaceae) in China

Phytotaxa ◽

10.11646/phytotaxa.414.3.1 ◽

2019 ◽

Vol 414 (3) ◽

pp. 121-128

Author(s):

RUI-JIANG WANG

Keyword(s):

New Combination ◽

Morphological Comparison ◽

Generic Delimitation ◽

New Synonyms ◽

Flora Of China

The genus Hedyotis sensu lato was splitted into several genera on basis of morphological and molecular evidences. Under this generic delimitation, seven new taxonomic treatments, including six new synonyms and one new combination, are proposed for the Flora of China. The name Hedyotis tenelliflora that misapplied to Scleromitrion angustifolium is also corrected with morphological comparison.

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Universal human papillomavirus typing by whole genome sequencing following target enrichment: evaluation of assay reproducibility and limit of detection

BMC Genomics ◽

10.1186/s12864-019-5598-0 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Tengguo Li ◽

Elizabeth R. Unger ◽

Mangalathu S. Rajeevan

Keyword(s):

Human Papillomavirus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Limit Of Detection ◽

Whole Genome ◽

Target Enrichment

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Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

BMC Genomics ◽

10.1186/1471-2164-14-856 ◽

2013 ◽

Vol 14 (1) ◽

pp. 856 ◽

Cited By ~ 15

Author(s):

Eva C Berglund ◽

Carl Lindqvist ◽

Shahina Hayat ◽

Elin Övernäs ◽

Niklas Henriksson ◽

...

Keyword(s):

Whole Genome ◽

Target Enrichment ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Accurate Detection

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Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma

Journal of Virology ◽

10.1128/jvi.00677-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 3

Author(s):

Katherine L. James ◽

Thushan I. de Silva ◽

Katherine Brown ◽

Hilton Whittle ◽

Stephen Taylor ◽

...

Keyword(s):

Genetic Diversity ◽

Blood Plasma ◽

De Novo ◽

A Priori ◽

Pcr Amplification ◽

Hiv Vaccine ◽

Whole Genome ◽

Plasma Samples ◽

Target Enrichment ◽

Rna Seq

ABSTRACTAccurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requiresa priorisequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i)de novogenome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory genevpxin all patients. Our study demonstrates that RNA-Seq is a feasible full-genomede novosequencing method for blood plasma samples collected from HIV-2-infected individuals.IMPORTANCEAn accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detaileda priorisequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.

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