Qualitative De Novo Analysis of Full Length cDNA and Quantitative Analysis of Gene Expression for Common Marmoset (Callithrix jacchus) Transcriptomes Using Parallel Long-Read Technology and Short-Read Sequencing

AbstractHigh-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-read sequencing is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. However, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here we introduce and validate a new long-read ONT based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single cell samples.Significance StatementSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behaviors in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The R2C2 method we are presenting here is the first method with sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single cell samples economically feasible.

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Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing

BMC Genomics ◽

10.1186/s12864-017-3757-8 ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 89

Author(s):

Nam V. Hoang ◽

Agnelo Furtado ◽

Patrick J. Mason ◽

Annelie Marquardt ◽

Lakshmi Kasirajan ◽

...

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Full Length ◽

Short Read ◽

Short Read Sequencing ◽

Sugarcane Genome

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Reference Plasmid pHXB2_D is an HIV-1 Molecular Clone that Exhibits Identical LTRs and a Single Integration Site Indicative of an HIV Provirus

10.21203/rs.3.rs-382311/v1 ◽

2021 ◽

Author(s):

Alejandro R. Gener ◽

Wei Zou ◽

Brian T. Foley ◽

Deborah P. Hyink ◽

Paul E. Klotman

Keyword(s):

Dna Sequencing ◽

De Novo ◽

Integration Site ◽

Full Length ◽

Long Terminal Repeats ◽

Single Nucleotide Variants ◽

Short Read ◽

Long Read ◽

Reference Plasmid ◽

Hiv 1

Abstract Objective: To compare long-read nanopore DNA sequencing (DNA-seq) with short-read sequencing-by-synthesis for sequencing a full-length (e.g., non-deletion, nor reporter) HIV-1 model provirus in plasmid pHXB2_D. Design: We sequenced pHXB2_D and a control plasmid pNL4-3_gag-pol(Δ1443-4553)_EGFP with long- and short-read DNA-seq, evaluating sample variability with resequencing (sequencing and mapping to reference HXB2) and de novo viral genome assembly. Methods: We prepared pHXB2_D and pNL4-3_gag-pol(Δ1443-4553)_EGFP for long-read nanopore DNA-seq, varying DNA polymerases Taq (Sigma-Aldrich) and Long Amplicon (LA) Taq (Takara). Nanopore basecallers were compared. After aligning reads to the reference HXB2 to evaluate sample coverage, we looked for variants. We next assembled reads into contigs, followed by finishing and polishing. We hired an external core to sequence-verify pHXB2_D and pNL4-3_gag-pol(Δ1443-4553)_EGFP with single-end 150 base-long Illumina reads, after masking sample identity. Results: We achieved full-coverage (100%) of HXB2 HIV-1 from 5' to 3' long terminal repeats (LTRs), with median per-base coverage of over 9000x in one experiment on a single MinION flow cell. The longest HIV-spanning read to-date was generated, at a length of 11,487 bases, which included full-length HIV-1 and plasmid backbone with flanking host sequences supporting a single HXB2 integration event. We discovered 20 single nucleotide variants in pHXB2_D compared to reference, verified by short-read DNA sequencing. There were no variants detected in the HIV-1 segments of pNL4-3_gag-pol(Δ1443-4553)_EGFP. Conclusions: Nanopore sequencing performed as-expected, phasing LTRs, and even covering full-length HIV. The discovery of variants in a reference plasmid demonstrates the need for sequence verification moving forward, in line with calls from funding agencies for reagent verification. These results illustrate the utility of long-read DNA-seq to advance the study of HIV at single integration site resolution.

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Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806447115 ◽

2018 ◽

Vol 115 (39) ◽

pp. 9726-9731 ◽

Cited By ~ 65

Author(s):

Roger Volden ◽

Theron Palmer ◽

Ashley Byrne ◽

Charles Cole ◽

Robert J. Schmitz ◽

...

Keyword(s):

Single Cell ◽

Full Length ◽

Long Distance ◽

Distance Information ◽

Short Read ◽

Transcript Isoforms ◽

Short Read Sequencing ◽

Sequencing Method ◽

Long Read ◽

Rna Transcript

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.

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Analysis of gene expression for microminipig liver transcriptomes using parallel long-read technology and short-read sequencing

Biopharmaceutics & Drug Disposition ◽

10.1002/bdd.2007 ◽

2016 ◽

Vol 37 (4) ◽

pp. 220-232 ◽

Cited By ~ 4

Author(s):

Chizuka Sakai ◽

Shunsuke Iwano ◽

Makiko Shimizu ◽

Jun Onodera ◽

Masashi Uchida ◽

...

Keyword(s):

Gene Expression ◽

Short Read ◽

Short Read Sequencing ◽

Long Read

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Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BMC Microbiology ◽

10.1186/s12866-021-02094-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yoshiyuki Matsuo ◽

Shinnosuke Komiya ◽

Yoshiaki Yasumizu ◽

Yuki Yasuoka ◽

Katsura Mizushima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

Short Read ◽

Short Read Sequencing ◽

Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

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High-Quality de novo Chromosome-Level Genome Assembly of a Single Bombyx mori With BmNPV Resistance by a Combination of PacBio Long-Read Sequencing, Illumina Short-Read Sequencing, and Hi-C Sequencing

Frontiers in Genetics ◽

10.3389/fgene.2021.718266 ◽

2021 ◽

Vol 12 ◽

Author(s):

Min Tang ◽

Suqun He ◽

Xun Gong ◽

Peng Lü ◽

Rehab H. Taha ◽

...

Keyword(s):

Bombyx Mori ◽

Genome Assembly ◽

De Novo ◽

High Quality ◽

Single Strain ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Reference Genomes ◽

Chromosome Level

The reference genomes of Bombyx mori (B. mori), Silkworm Knowledge-based database (SilkDB) and SilkBase, have served as the gold standard for nearly two decades. Their use has fundamentally shaped model organisms and accelerated relevant studies on lepidoptera. However, the current reference genomes of B. mori do not accurately represent the full set of genes for any single strain. As new genome-wide sequencing technologies have emerged and the cost of high-throughput sequencing technology has fallen, it is now possible for standard laboratories to perform full-genome assembly for specific strains. Here we present a high-quality de novo chromosome-level genome assembly of a single B. mori with nuclear polyhedrosis virus (BmNPV) resistance through the integration of PacBio long-read sequencing, Illumina short-read sequencing, and Hi-C sequencing. In addition, regular bioinformatics analyses, such as gene family, phylogenetic, and divergence analyses, were performed. The sample was from our unique B. mori species (NB), which has strong inborn resistance to BmNPV. Our genome assembly showed good collinearity with SilkDB and SilkBase and particular regions. To the best of our knowledge, this is the first genome assembly with BmNPV resistance, which should be a more accurate insect model for resistance studies.

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scCAT-seq:single-cell identification and quantification of mRNA isoforms by cost-effective short-read sequencing of cap and tail

10.1101/2019.12.11.873505 ◽

2019 ◽

Author(s):

Youjin Hu ◽

Jiawei Zhong ◽

Yuhua Xiao ◽

Zheng Xing ◽

Katherine Sheu ◽

...

Keyword(s):

Single Cell ◽

Learning Algorithm ◽

Single Cells ◽

Full Length ◽

Translation Efficiency ◽

Mrna Isoforms ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Identification And Quantification

AbstractThe differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Isoforms also allow a single gene different functions across various tissues and cells However, methods for efficient genome-wide identification and quantification of RNA isoforms in single cells are still lacking. Here, we introduce single cell Cap And Tail sequencing (scCAT-seq). In conjunction with a novel machine learning algorithm developed for TSS/TES characterization, scCAT-seq can demarcate transcript boundaries of RNA transcripts, providing an unprecedented way to identify and quantify single-cell full-length RNA isoforms based on short-read sequencing. Compared with existing long-read sequencing methods, scCAT-seq has higher efficiency with lower cost. Using scCAT-seq, we identified hundreds of previously uncharacterized full-length transcripts and thousands of alternative transcripts for known genes, quantitatively revealed cell-type specific isoforms with alternative TSSs/TESs in dorsal root ganglion (DRG) neurons, mature oocytes and ageing oocytes, and generated the first atlas of the non-human primate cornea. The approach described here can be widely adapted to other short-read or long-read methods to improve accuracy and efficiency in assessing RNA isoform dynamics among single cells.

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Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

10.1101/2020.05.06.078147 ◽

2020 ◽

Cited By ~ 3

Author(s):

Yoshiyuki Matsuo ◽

Shinnosuke Komiya ◽

Yoshiaki Yasumizu ◽

Yuki Yasuoka ◽

Katsura Mizushima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

Short Read ◽

Short Read Sequencing ◽

16S Amplicon Sequencing ◽

Long Read

AbstractBackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

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