scholarly journals Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

2019 ◽  
Author(s):  
Søren M. Karst ◽  
Ryan M. Ziels ◽  
Rasmus H. Kirkegaard ◽  
Emil A. Sørensen ◽  
Daniel McDonald ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Amplicon Sequencing ◽  
High Accuracy ◽  
Pacbio Sequencing ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genomic Regions ◽  
Oxford Nanopore Technologies

AbstractHigh-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.

scholarly journals Full-coverage sequencing of HIV-1 provirus from a reference plasmid

2019 ◽  
Author(s):  
Alejandro R. Gener
Keyword(s):  
Single Molecule ◽  
Integration Site ◽  
Full Length ◽  
Rapid Pcr ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Plasmid ◽  
Nanopore Dna Sequencing ◽  
Oxford Nanopore Technologies ◽  
Hiv 1

ABSTRACTObjective(s)To evaluate nanopore DNA sequencing for sequencing full-length HIV-1 provirus.DesignI used nanopore sequencing to sequence full-length HIV-1 from a plasmid (pHXB2).MethodspHXB2 plasmid was processed with the Rapid PCR-Barcoding library kit and sequenced on the MinION sequencer (Oxford Nanopore Technologies, Oxford., UK). Raw fast5 reads were converted into fastq (base called) with Albacore, Guppy, and FlipFlop base callers. Reads were first aligned to the reference with BWA-MEM to evaluate sample coverage manually. Reads were then assembled with Canu into contigs, and contigs manually finished in SnapGene.ResultsI sequenced full-length HXB2 HIV-1 from 5’ to 3’ LTR (100%), with median per-base coverage of over 9000x in one 12-barcoded 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 on either side. At least 20 variants were discovered in pHXB2 compared to reference.ConclusionsThe MinION sequencer performed as-expected, covering full-length HIV. The discovery of variants in a dogmatic reference plasmid demonstrates the need for single-molecule sequence verification moving forward. These results illustrate the utility of long read sequencing to advance the study of HIV at single integration site resolution.

scholarly journals Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation

2016 ◽  
Author(s):  
Sergey Koren ◽  
Brian P. Walenz ◽  
Konstantin Berlin ◽  
Jason R. Miller ◽  
Nicholas H. Bergman ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Error Rates ◽  
Celera Assembler ◽  
Oxford Nanopore ◽  
Long Read ◽  
Reference Quality ◽  
Order Of Magnitude ◽  
Assembly Algorithms ◽  
Oxford Nanopore Technologies

AbstractLong-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

scholarly journals Deciphering Neurodegenerative Diseases Using Long-Read Sequencing

Neurology ◽  
2021 ◽  
pp. 10.1212/WNL.0000000000012466
Author(s):  
Yun Su ◽  
Liyuan Fan ◽  
Changhe Shi ◽  
Tai Wang ◽  
Huimin Zheng ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Single Molecule ◽  
Direct Detection ◽  
Gc Content ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Repeat Expansions ◽  
Genomic Regions

Neurodegenerative diseases exhibit chronic progressive lesions in the central and peripheral nervous systems with unclear causes. The search for pathogenic mutations in human neurodegenerative diseases has benefited from massively parallel short-read sequencers. However, genomic regions, including repetitive elements, especially with high/low GC content, are far beyond the capability of conventional approaches. Recently, long-read single-molecule DNA sequencing technologies have emerged and enabled researchers to study genomes, transcriptomes, and metagenomes at unprecedented resolutions. The identification of novel mutations in unresolved neurodegenerative disorders, the characterization of causative repeat expansions, and the direct detection of epigenetic modifications on naive DNA by virtue of long-read sequencers will further expand our understanding of neurodegenerative diseases. In this paper, we review and compare two prevailing long-read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), and discuss their applications in neurodegenerative diseases.

scholarly journals HIT-scISOseq: High-throughput and High-accuracy Single-cell Full-length Isoform Sequencing for Corneal Epithelium

2020 ◽  
Author(s):  
Ying-Feng Zheng ◽  
Zhi-Chao Chen ◽  
Zhuo-Xing Shi ◽  
Kun-Hua Hu ◽  
Jia-Yong Zhong ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Cost Effective ◽  
Pcr Primers ◽  
Cell Types ◽  
High Accuracy ◽  
Full Length ◽  
Pacbio Sequencing ◽  
Simple Method ◽  
Long Read

AbstractSingle-cell isoform sequencing can reveal transcriptomic dynamics in individual cells invisible to bulk- and single-cell RNA analysis based on short-read sequencing. However, current long-read single-cell sequencing technologies have been limited by low throughput and high error rate. Here we introduce HIT-scISOseq for high-throughput single-cell isoform sequencing. This method was made possible by full-length cDNA capture using biotinylated PCR primers, and by our novel library preparation procedure that combines head-to-tail concatemeric full-length cDNAs into a long SMRTbell insert for high-accuracy PacBio sequencing. HIT-scISOseq yields > 10 million high-accuracy full-length isoforms in a single PacBio Sequel II 8M SMRT Cell, providing > 8 times more data output than the standard single-cell isoform PacBio sequencing protocol. We exemplified HIT-scISOseq by first studying transcriptome profiles of 4,000 normal and 8,000 injured corneal epitheliums from cynomolgus monkeys. We constructed dynamic transcriptome landscapes of known and rare cell types, revealed novel isoforms, and identified injury-related splicing and switching events that are previously not accessible with low throughput isoform sequencing. HIT-scISOseq represents a high-throughput, cost-effective, and technically simple method to accelerate the burgeoning field of long-read single-cell transcriptomics.

scholarly journals Combined nanopore and single-molecule real-time sequencing survey of human betaherpesvirus 5 transcriptome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Balázs Kakuk ◽  
Dóra Tombácz ◽  
Zsolt Balázs ◽  
Norbert Moldován ◽  
Zsolt Csabai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Splice Variants ◽  
Splice Isoforms ◽  
Short Read Sequencing ◽  
Novel Transcript ◽  
Novel Approach ◽  
Oxford Nanopore ◽  
Long Read ◽  
Polycistronic Transcripts ◽  
Oxford Nanopore Technologies

AbstractLong-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

scholarly journals Combined Nanopore and Single-Molecule Real-Time Sequencing Survey of Human Betaherpesvirus 5 Transcriptome

2021 ◽  
Author(s):  
Balazs Kakuk ◽  
Dora Tombacz ◽  
Zsolt Balazs ◽  
Norbert Moldovan ◽  
Zsolt Csabai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Splice Variants ◽  
Splice Isoforms ◽  
Short Read Sequencing ◽  
Novel Transcript ◽  
Novel Approach ◽  
Oxford Nanopore ◽  
Long Read ◽  
Polycistronic Transcripts ◽  
Oxford Nanopore Technologies

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

scholarly journals NGSpeciesID: DNA barcode and amplicon consensus generation from long-read sequencing data

2020 ◽  
Author(s):  
Kristoffer Sahlin ◽  
Marisa Lim ◽  
Stefan Prost
Keyword(s):  
High Throughput Sequencing ◽  
Dna Barcode ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Sequencing Data ◽  
Sequencing Platform ◽  
Consensus Sequences ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

Third generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have gained popularity over the last years. These platforms can generate millions of long read sequences. This is not only advantageous for genome sequencing projects, but also for amplicon-based high-throughput sequencing experiments, such as DNA barcoding. However, the relatively high error rates associated with these technologies still pose challenges for generating high quality consensus sequences. Here we present NGSpeciesID, a program which can generate highly accurate consensus sequences from long-read amplicon sequencing technologies, including ONT and PacBio. The tool includes clustering of the reads to help filter out contaminants or reads with high error rates and employs polishing strategies specific to the appropriate sequencing platform. We show that NGSpeciesID produces consensus sequences with improved usability by minimizing preprocessing and software installation and scalability by enabling rapid processing of hundreds to thousands of samples, while maintaining similar consensus accuracy as current pipelines

scholarly journals Amplicon_sorter: a tool for reference-free amplicon sorting based on sequence similarity and for building consensus sequences

2021 ◽  
Author(s):  
Andy Vierstraete ◽  
Bart Braeckman
Keyword(s):  
Sequence Similarity ◽  
Low Cost ◽  
Consensus Sequence ◽  
Consensus Sequences ◽  
Third Generation Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing

Oxford Nanopore Technologies (ONT) is a third-generation sequencing technology that is gaining popularity in ecological research for its portable and low-cost sequencing possibilities. Although the technology is primarily developed for long-read sequencing, it can also be applied to sequence amplicons. The downside of ONT is the low quality of the raw reads. Hence, generating a high-quality consensus sequence is still a challenge. We present Amplicon_sorter, a tool for reference-free sorting of ONT sequenced amplicons based on their similarity in sequence and length and for building solid consensus sequences.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

Dual Isoform Sequencing Reveals a Multifaceted Transcriptional Architecture of a Prototype Baculovirus

2021 ◽  
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Norbert Moldován ◽  
Ádám Fülöp ◽  
István Prazsák ◽  
...  
Keyword(s):  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Oxford Nanopore ◽  
The Pacific ◽  
Viral Genes ◽  
Long Read ◽  
Oxford Nanopore Technologies ◽  
Overlapping Transcripts

Abstract In this study, we used two long-read sequencing (LRS) techniques, Sequel from the Pacific Biosciences and MinION from Oxford Nanopore Technologies, for the transcriptional characterization of a prototype baculovirus, Autographacalifornica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby to distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcripts, of which 759 are novel and 116 have been annotated previously. These RNA molecules include 41 novel putative protein coding transcript (each containing 5’-truncated in-frame ORFs), 14 monocistronic transcripts, 99 multicistronic RNAs, 101 non-coding RNA, and 504 length isoforms. We also detected RNA methylation in 12 viral genes and RNA hyper-editing in the longer 5’-UTR transcript isoform of ORF 19 gene.

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