CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

Routh, Andrew; Chang, Max W.; Okulicz, Jason F.; Johnson, John E.; Torbett, Bruce E.

doi:10.1016/j.ymeth.2015.09.021

Cited by 19 publications

(26 citation statements)

References 29 publications

(38 reference statements)

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“…Because it can copy long RNA molecules end-to-end, the MarathonRT may be useful for characterizing heterogenous RNA populations when combined with third-generation end-to-end sequencing platforms (Chaisson et al 2015;Jain et al 2016). This would allow investigators to identify and quantify alternative splicing isoforms (Bolisetty et al 2015), correlated mutations in viral quasi-species (Wu et al 2014;Routh et al 2015), variation in RNA-editing among different transcripts (Levanon et al 2004;Meyer et al 2012), and variations in secondary structures (Siegfried et al 2014;Zubradt et al 2017).…”

Section: Discussionmentioning

confidence: 99%

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

Zhao

Liu

Pyle

2017

RNA

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Group II introns and non-LTR retrotransposons encode a phylogenetically related family of highly processive reverse transcriptases (RTs) that are essential for mobility and persistence of these retroelements. Recent crystallographic studies on members of this RT family have revealed that they are structurally distinct from the retroviral RTs that are typically used in biotechnology. However, quantitative, structure-guided analysis of processivity, efficiency, and accuracy of this alternate RT family has been lacking. Here, we characterize the processivity of a group II intron maturase RT from Eubacterium rectale (E.r.), for which high-resolution structural information is available. We find that the E.r. maturase RT (MarathonRT) efficiently copies transcripts at least 10 kb in length and displays superior intrinsic RT processivity compared to commercial enzymes such as Superscript IV (SSIV). The elevated processivity of MarathonRT is at least partly mediated by a loop structure in the finger subdomain that acts as a steric guard (the α-loop). Additionally, we find that a positively charged secondary RNA binding site on the surface of the RT diminishes the primer utilization efficiency of the enzyme, and that reengineering of this surface enhances capabilities of the MarathonRT. Finally, using single-molecule sequencing, we show that the error frequency of MarathonRT is comparable to that of other high-performance RTs, such as SSIV, which were tested in parallel. Our results provide a structural framework for understanding the enhanced processivity of retroelement RTs, and they demonstrate the potential for engineering a powerful new generation of RT tools for application in biotechnology and research.

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Section: Discussionmentioning

confidence: 99%

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

Zhao

Liu

Pyle

2017

RNA

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“…Co-Variation Mapper was used to identify correlated amino acid variants found in high prevalence among HA sequences at the sub-consensus level [44]. To achieve that, filtered reads were aligned with the HA sequence of the reference virus using more stringent alignment options in BWA allowing a maximum of 1 mismatch per aligned read.…”

Section: Haplotype Reconstruction and Diversity Analysismentioning

confidence: 99%

“…To achieve that, filtered reads were aligned with the HA sequence of the reference virus using more stringent alignment options in BWA allowing a maximum of 1 mismatch per aligned read. The output SAM files were then passed to the Co-VaMa scripts to find evidence of linkage disequilibrium (LD) [44]. Resulting LD mean values for each mutation pair were then evaluated, and association was confirmed for any mutation pair with LD values that exceeded the 3σ significance limit to ensure high confidence.…”

Section: Haplotype Reconstruction and Diversity Analysismentioning

confidence: 99%

Inter-Versus Intra-Host Sequence Diversity of pH1N1 and Associated Clinical Outcomes

et al. 2020

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The diversity of RNA viruses dictates their evolution in a particular host, community or environment. Here, we reported within- and between-host pH1N1virus diversity at consensus and sub-consensus levels over a three-year period (2015–2017) and its implications on disease severity. A total of 90 nasal samples positive for the pH1N1 virus were deep-sequenced and analyzed to detect low-frequency variants (LFVs) and haplotypes. Parallel evolution of LFVs was seen in the hemagglutinin (HA) gene across three scales: among patients (33%), across years (22%), and at global scale. Remarkably, investigating the emergence of LFVs at the consensus level demonstrated that within-host virus evolution recapitulates evolutionary dynamics seen at the global scale. Analysis of virus diversity at the HA haplotype level revealed the clustering of low-frequency haplotypes from early 2015 with dominant strains of 2016, indicating rapid haplotype evolution. Haplotype sharing was also noticed in all years, strongly suggesting haplotype transmission among patients infected during a specific influenza season. Finally, more than half of patients with severe symptoms harbored a larger number of haplotypes, mostly in patients under the age of five. Therefore, patient age, haplotype diversity, and the presence of certain LFVs should be considered when interpreting illness severity. In addition to its importance in understanding virus evolution, sub-consensus virus diversity together with whole genome sequencing is essential to explain variabilities in clinical outcomes that cannot be explained by either analysis alone.

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“…Importantly, these ssDNA templates are bio-compatible (50). Therefore, with a standard PCR reaction we can amplify these fragments to generate high-quality Illumina sequencing libraries with even sequence coverage (51).…”

Section: Poly(a)-clickseq Library Generationmentioning

confidence: 99%

Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

Routh

Jaworski

et al. 2017

Preprint

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The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture, and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3´ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error, and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called "ClickSeq". Here, we adapt this method to direct the cDNA synthesis specifically toward the 3´UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PACseq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

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CoVaMa: Co-Variation Mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data

Cited by 19 publications

References 29 publications

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

Inter-Versus Intra-Host Sequence Diversity of pH1N1 and Associated Clinical Outcomes

Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

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