Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

Wu, Douglas C.; Lambowitz, Alan M.

doi:10.1038/s41598-017-09064-w

Cited by 31 publications

(52 citation statements)

References 61 publications

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“…Analysis of TGIRT-seq datasets obtained for fragmented UHRs or plasma DNA suggested that a major source of sequence bias is the DNA ligation step using the thermostable 5' App DNA/RNA ligase, which has a preference for A or C and against U/T at position -3 from the 3' end of the acceptor nucleic acid (Jackson et al 2014;Nottingham et al 2016;Wu and Lambowitz 2017). We noticed that the R2R adapter used currently for TGIRT-seq (denoted NTC based on its 3' end sequence) has a C-residue at position -3 from its 3' end, which favors the formation of R1R-R2R adapter dimers during the ligation step ( Fig.…”

Section: A Single Nucleotide Change In the R2r Adapter Strongly Decrementioning

confidence: 99%

“…Here, we used the previously determined ligation biases of the thermostable 5' App RNA/DNA ligase (Jackson et al 2014;Nottingham et al, 2016;Wu and Lambowitz 2017) to design an R2/R2R adapter with just a single nucleotide change that dramatically decreases the formation of adapter dimers, thereby improving the recovery of miRNA sequences and enabling the construction of TGIRT-seq libraries from smaller amounts of starting material. Additionally, using a miRNA reference set containing an equimolar mix of 962 human miRNAs, we systematically analyzed 5'-and 3'-end biases in TGIRT-seq introduced by the thermostable 5' App RNA/DNA ligase and template switching, respectively, and developed biochemical and computational methods for ameliorating these biases.…”

Section: Introductionmentioning

confidence: 99%

“…Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter byusing 5' App DNA/RNA ligase (New England Biolabs), resulting in formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. (B)Taking into account known biases of the 5' App DNA/RNA ligase(Jackson et al 2014;Nottingham et al 2016;Wu and Lambowitz, 2017), the R2R adapter used previously in TGIRTseq (denoted NTC) was modified by inserting a single T-residue at position -3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. (C)Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters.…”

mentioning

confidence: 99%

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Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Yao

et al. 2018

Preprint

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Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNAseq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5'and 3'-end biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5'-and 3'-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.

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Section: A Single Nucleotide Change In the R2r Adapter Strongly Decrementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Yao

et al. 2018

Preprint

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“…18 However, the current ssDNA-based methods are still restricted by some limitations such as input DNA dephosphorylation, isolation with biotin-magnetic beads and dependence on special ligase (e.g. CircLigase II) 15,17,18 or retroviral reverse transcriptase (e.g. DNA SMART ChIP-seq Kit, Clontech).…”

Section: Introductionmentioning

confidence: 99%

Decoding genetic and epigenetic information embedded in cell free DNA with adapted SALP‐seq

Dai

et al. 2019

Intl Journal of Cancer

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Cell free DNA (cfDNA) in human plasma carries abundant information, which has therefore been the key sample for noninvasive prenatal testing (NIPT) and liquid biopsy. Especially by using the rapidly developed next‐generation sequencing (NGS) techniques, the genetic and epigenetic information embedded in cfDNA has been effectively and extensively decoded. In this process, a key step is to construct the NGS library. Due to its high degradation, the single strand‐based method was reported to be more qualified to construct the NGS library of cfDNA. In order to develop a new simple method for this application, this study adapted our recently developed single strand adaptor library preparation (SALP) method for constructing an NGS library of cfDNA. In the improved method, cfDNA was firstly denatured into single strands and then ligated with a single strand adaptor (SSA) that had a 3′ overhang of 3 random bases by using T4 DNA ligase. The SSA‐ligated DNA was converted into double‐stranded DNA with an additional adenine at the other end by polymerizing with Taq polymerase. Next, a barcode T adaptor (BTA) was ligated to this end. Finally, the cfDNA ligated with two adaptors was amplified with the Illumina‐compatible primers for NGS. Using the method, this study successfully sequenced 20 cfDNA samples from 16 esophageal cancer patients and 4 healthy people. By bioinformatics analysis, this study found the genetic and epigenetic difference between patients and healthy people and identified 23 epigenetic and 28 genetic altered esophageal cancer‐specific genes.

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“…Unlike the R2 element RT (33,34), group II intron RTs do not need to be actively elongating to template switch and can do so directly from synthetic RNA template/DNA primer substrates with a single-nucleotide (1-nt) 3'-DNA overhang that base pairs with the 3' nucleotide of an RNA acceptor (6). This reaction enables the precise attachment of 3' RNA-seq adapters to target RNAs or DNAs in a single step without tailing or ligation (8,9,40).…”

Section: Introductionmentioning

confidence: 99%

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

Yao

Russell

et al. 2019

Preprint

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Group II intron reverse transcriptase template switching 2 ABSTRACTThe reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retroelements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. The reliance on a single base pair with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTRretroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

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Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

Cited by 31 publications

References 61 publications

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

Decoding genetic and epigenetic information embedded in cell free DNA with adapted SALP‐seq

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

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