Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes

Shin, Seung Chul; Ahn, Do Hwan; Kim, Su Jin; Lee, Hyoungseok; Oh, Tae‐Jin; Lee, Jong Eun; Park, Hyun

doi:10.1371/journal.pone.0068824

Cited by 70 publications

(50 citation statements)

References 15 publications

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“…This result verifies the findings that repeated sequence in the genome induces complexity and poses the greatest challenge to all assembly algorithms [17]. Therefore, consistent with [22, 20], we suggest that the simplest and most effective way to produce a de novo GC-rich bacterial genome assembly is with PacBio RS II long-reads using HGAP 2.0 assembler to self-correct the reads. Further, we were able to complete a reference genome by using only one SMRT cell.…”

Section: Discussionsupporting

confidence: 89%

Comparison of Genome Sequencing Technology and Assembly Methods for the Analysis of a GC-Rich Bacterial Genome

Scott

Ely

2014

Curr Microbiol

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Motivation Improvements in technology and decreases in price have made de novo bacterial genomic sequencing a reality for many researchers, but it has created a need to evaluate the methods for generating a complete and accurate genome assembly. Results We sequenced the GC-rich Caulobacter henricii genome using the Illumina MiSeq, Roche 454, and Pacific Biosciences RS II sequencing systems. To generate a complete genome sequence, we performed assemblies using eight readily available programs and found that builds using the Illumina MiSeq and the Roche 454 data produced accurate yet numerous contigs. SPAdes performed the best followed by PANDAseq. In contrast, the Celera Assembler produced a single genomic contig using the Pacific Biosciences data after error correction with the Illumina MiSeq data. In addition, we duplicated this build using the Pacific Biosciences data with HGAP2.0. The accuracy of these builds was verified by Pulsed Field Gel Electrophoresis of genomic DNA cut with restriction enzymes.

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Section: Discussionsupporting

confidence: 89%

Comparison of Genome Sequencing Technology and Assembly Methods for the Analysis of a GC-Rich Bacterial Genome

Scott

Ely

2014

Curr Microbiol

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“…The advantages of including longer PacBio reads, e.g. hybrid assembly or PacBio reads combined with optical mapping, were also demonstrated by previous studies in the sequencing of bacterial and fungal genomes5152. Especially for small genome, near-gapless even completely finished genome could be obtained5354.…”

Section: Discussionmentioning

confidence: 90%

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

Yin

et al. 2017

Sci Rep

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Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.

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“…Applied to the sequencing of PCR products, PacBio reads maintain the entire product as an uninterrupted sequence, allowing reliable identification against reference libraries with levels of similarity equivalent to those of HERV-Ks (7). A comprehensive description of the features and advantages of this technology were published previously (33,34).…”

Section: Resultsmentioning

confidence: 99%

HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes

Brinzevich

Young²,

Sebra

et al. 2014

J Virol

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Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) singlemolecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication. IMPORTANCEHere, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deepsequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins retain their ability to make a protein. Importantly, one of them, the envelope glycoprotein of HERV-K18, is incorporated into HIV-1 in an HIV matrix-specific fashion. The ramifications of such interactions are discussed, as the possibility of HIV-1 target tissue broadening and immune evasion are considered.

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Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes

Cited by 70 publications

References 15 publications

Comparison of Genome Sequencing Technology and Assembly Methods for the Analysis of a GC-Rich Bacterial Genome

Comparison of Genome Sequencing Technology and Assembly Methods for the Analysis of a GC-Rich Bacterial Genome

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes

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