ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments

Schlecht, Ulrich; Mok, Janine; Dallett, Carolina; Berka, Jan

doi:10.1038/s41598-017-05503-w

Cited by 17 publications

(35 citation statements)

References 31 publications

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“…The strategy of SMURF-seq is to concatenate short fragments into very long molecules (∼8 kb) prior to sequencing. The concept of ligating short molecules together prior to sequencing was introduced in serial analysis of gene expression (SAGE) [9] and then subsequently used in short multiply aggregated sequence homologies (SMASH) for CNV profiling using Illumina short-read technology [10] and ConcatSeq for target enrichment workflows on PacBio machines [11]. SMURF-seq differs from these methods in that the fragmented and re-ligated molecules are substantially longer, it allows for variable fragment lengths as permitted by long-read sequencing, and the number of fragments within each read is substantially greater.…”

Section: Introductionmentioning

confidence: 99%

SMURF-seq: efficient copy number profiling on long-read sequencers

Prabakar

Hicks

et al. 2019

Genome Biol

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We present SMURF-seq, a protocol to efficiently sequence short DNA molecules on a long-read sequencer by randomly ligating them to form long molecules. Applying SMURF-seq using the Oxford Nanopore MinION yields up to 30 fragments per read, providing an average of 6.2 and up to 7.5 million mappable fragments per run, increasing information throughput for read-counting applications. We apply SMURF-seq on the MinION to generate copy number profiles. A comparison with profiles from Illumina sequencing reveals that SMURF-seq attains similar accuracy. More broadly, SMURF-seq expands the utility of long-read sequencers for read-counting applications. Electronic supplementary material The online version of this article (10.1186/s13059-019-1732-1) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

SMURF-seq: efficient copy number profiling on long-read sequencers

Prabakar

Hicks

et al. 2019

Genome Biol

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“… Methods to improve the sequencing yield and the basecalling accuracy past the specifications of the long-read sequencing platforms. The methods presented are the ConcatSeq ( Schlecht et al, 2017 ), INC-seq ( Li et al, 2016 ), R2C2 ( Volden et al, 2018 ), and the PacBio Circular Consensus Sequencing. In the section “increase in yield,” the green boxes present at either the 5′ end or the 3′ end, depending on the pool of the cDNA molecules, indicate the same DNA sequence.…”

Section: Library Preparation Methodologies To Decrease the Error Ratementioning

confidence: 99%

“…Re-engineering the ZMW microwells by introducing nanopores at the bottom has been proposed to alleviate this problem and offer a complete saturation of the SMRT cell (Larkin et al, 2017). Nevertheless, without re-engineering the SMRT cell, there have been studies that are exploiting the concatemerisation in the form of the ConcatSeq (Schlecht et al, 2017) protocol, where the same cDNA molecules are amplified in two separate reactions with two distinct sets of primers. The one primer set has complementary ends to the other primer set and after pooling the two cDNA populations together, a Gibson assembly reaction is followed where the ends of the two cDNA populations are recombining together, resulting in concatamers of the individual cDNA molecules (Figure 2).…”

Section: Toward Increasing the Number Of Sequenced Molecules Beyond Tmentioning

confidence: 99%

Methodologies for Transcript Profiling Using Long-Read Technologies

et al. 2020

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RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale highthroughput studies. NGS technologies comprise high throughput, cost efficient shortread RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward longread sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5 and 3 UTRs is still in development.

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“…This is especially problematic since high coverage is needed to compensate for the higher error rate of Nanopore generated sequences. A promising approach to overcome these limitations is the concatenation of PCR amplicons that allows the sequencing of several amplicons within a single read, thereby exponentially increasing throughput for genotyping (Cornelis et al 2017;Schlecht et al 2017) .…”

Section: Introductionmentioning

confidence: 99%

“…Here, we report on the development and performance of a novel Nanopore-based in-field SNP GSI method by adapting existing SNP GSI technology to the Nanopore platform using a concatenation approach (Schlecht et al 2017). We aim to demonstrate in-field feasibility, repeatability and comparability to established platforms.…”

Section: Introductionmentioning

confidence: 99%

In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment

Sutherland

et al. 2021

Preprint

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Genetic stock identification (GSI) by single nucleotide polymorphism (SNP) sequencing has become the gold standard for stock identification in Pacific salmon, which are found in mixed-stocks during the oceanic phase of their lifecycle. Sequencing platforms currently applied require large batch sizes and multi-day processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, like the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and hold promise for the application of real-time, in-field stock identification on variable batch sizes. Here we report and evaluate utility and comparability of at-sea stock identification of coho salmon Oncorhynchus kisutch based on targeted SNP amplicon sequencing on the minION platform during the International Year of the Salmon Signature Expedition to the Gulf of Alaska in the winter of 2019. Long read sequencers are not optimized for short amplicons, therefore we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded stock assignment for 50 of the 80 assessed individuals. Nanopore-based SNP calls agreed with Ion Torrent based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed coho salmon in the queried baseline dataset contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time, accuracy, throughput, and cost, as well as augmentation of the existing baselines, specifically in stocks from coastal northern BC and Alaska. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach. Genotyping by amplicon sequencing in the hands of diverse stakeholders could inform management decisions over a broad expanse of the coast by allowing the analysis of small batches in remote areas in near real-time.

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ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments

Cited by 17 publications

References 31 publications

SMURF-seq: efficient copy number profiling on long-read sequencers

SMURF-seq: efficient copy number profiling on long-read sequencers

Methodologies for Transcript Profiling Using Long-Read Technologies

In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment

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