High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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Episo: quantitative estimation of RNA 5-methylcytosine at isoform level by high-throughput sequencing of RNA treated with bisulfite

Bioinformatics ◽

10.1093/bioinformatics/btz900 ◽

2019 ◽

Vol 36 (7) ◽

pp. 2033-2039 ◽

Cited By ~ 2

Author(s):

Junfeng Liu ◽

Ziyang An ◽

Jianjun Luo ◽

Jing Li ◽

Feifei Li ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Quantitative Estimation ◽

Supplementary Information ◽

Biological Processes ◽

Single Nucleotide ◽

Rna Immunoprecipitation ◽

Nucleotide Resolution ◽

Human And Mouse ◽

Single Nucleotide Resolution

Abstract Motivation RNA 5-methylcytosine (m5C) is a type of post-transcriptional modification that may be involved in numerous biological processes and tumorigenesis. RNA m5C can be profiled at single-nucleotide resolution by high-throughput sequencing of RNA treated with bisulfite (RNA-BisSeq). However, the exploration of transcriptome-wide profile and potential function of m5C in splicing remains to be elucidated due to lack of isoform level m5C quantification tool. Results We developed a computational package to quantify Epitranscriptomal RNA m5C at the transcript isoform level (named Episo). Episo consists of three tools: mapper, quant and Bisulfitefq, for mapping, quantifying and simulating RNA-BisSeq data, respectively. The high accuracy of Episo was validated using an improved m5C-specific methylated RNA immunoprecipitation (meRIP) protocol, as well as a set of in silico experiments. By applying Episo to public human and mouse RNA-BisSeq data, we found that the RNA m5C is not evenly distributed among the transcript isoforms, implying the m5C may subject to be regulated at isoform level. Availability and implementation Episo is released under the GNU GPLv3+ license. The resource code Episo is freely accessible from https://github.com/liujunfengtop/Episo (with Tophat/cufflink) and https://github.com/liujunfengtop/Episo/tree/master/Episo_Kallisto (with Kallisto). Supplementary information Supplementary data are available at Bioinformatics online.

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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

10.1101/392332 ◽

2018 ◽

Cited By ~ 5

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate.In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowedE. colistrains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in severalE. colistrains.There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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DNAscent v2: detecting replication forks in nanopore sequencing data with deep learning

BMC Genomics ◽

10.1186/s12864-021-07736-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Michael A. Boemo

Keyword(s):

Dna Replication ◽

High Throughput ◽

Single Molecule ◽

Replication Fork ◽

Genome Stability ◽

Brdu Incorporation ◽

Sequencing Data ◽

Single Nucleotide ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

Abstract Background Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software. Results This paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at https://github.com/MBoemo/DNAscent. Conclusions This paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.

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