Evaluation of tools for long read RNA-seq splice-aware alignment

Križanović, Krešimir; Echchiki, Amina; Roux, Julien; Šikić, Mile

doi:10.1093/bioinformatics/btx668

Cited by 67 publications

(64 citation statements)

References 20 publications

(19 reference statements)

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“…Conventional "RNA-seq" with short reads, which is rather inappropriately termed so since the RNA molecules are not directly sequenced (Hrdlickova, Toloue, & Tian, 2017), requires reverse transcription (RT) of the template RNA molecule into complementary DNA (cDNA), which is typically further amplified by PCR. The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018). The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018).…”

Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

“…Direct RNA sequencing (again, it is unfortunate that true RNA sequencing has to add the word "direct" to differentiate itself from conventional "RNA-Seq"), on the other hand, directly detects the ribonucleobases passing through the pore without RT or PCR amplification, and this approach is free of possible biases or misamplifications introduced during such steps. The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018). Such an application is also practical for the study of RNA viral genomes, because the genome has multiple reading frames, anti-sense gene locations, inefficient termination signals, and complex splice forms, and gene annotation is challenging using the conventional RNA-seq method (Depledge et al, 2018).…”

Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

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Nanopore sequencing: Review of potential applications in functional genomics

2019

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Molecular biology has been led by various measurement technologies, and increased throughput has developed omics analysis. The development of massively parallel sequencing technology has enabled access to fundamental molecular data and revealed genomic and transcriptomic signatures. Nanopore sequencers have driven such evolution to the next stage. Oxford Nanopore Technologies Inc. provides a new type of single molecule sequencer using protein nanopore that realizes direct sequencing without DNA synthesizing or amplification. This nanopore sequencer can sequence an ultra‐long read limited by the input nucleotide length, or can determine DNA/RNA modifications. Recently, many fields such as medicine, epidemiology, ecology, and education have benefited from this technology. In this review, we explain the features and functions of the nanopore sequencer, introduce various situations where it has been used as a critical technology, and expected future applications.

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Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

Section: Tr Anscrip Tome Analys Is and D Irec T Rna S Equen Cingmentioning

confidence: 99%

Nanopore sequencing: Review of potential applications in functional genomics

2019

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“…Then high (30X) and low (4X) coverage simulations were implemented for the highly and the lowly expressed isoforms, respectively, and the simulated reads were mixed to build the dataset. To benchmark the aligners on the datasets from various species and in various coverages more comprehensively, we referred to a previous study [30] to randomly select three sets of genes respectively from the three species to simulate 9 datasets. Each of the datasets was built with one of the gene-sets and a fixed coverage (4X, 10X or 30X).…”

Section: Results On Simulated Datasetsmentioning

confidence: 99%

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Liu

Zang

et al. 2019

Preprint

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Long-read RNA sequencing (RNA-seq) is promising to transcriptomics studies, however, the alignment of long RNA-seq reads is still non-trivial due to high sequencing errors and complicated gene structures. Herein, we propose deSALT, a tailored two-pass alignment approach, which constructs graph-based alignment skeletons to infer exons and uses them to generate spliced reference sequences to produce refined alignments. deSALT addresses several difficult technical issues, such as small exons and sequencing errors, which breakthroughs the bottlenecks of long RNA-seq read alignment. Benchmarks demonstrate that deSALT has a greater ability to produce accurate and homogeneous full-length alignments. deSALT is available at: https://github.com/hitbc/deSALT. Keywords long read alignment, RNA-seq, de Bruijn graph-based index 3 Background RNA sequencing (RNA-seq) has become a fundamental approach to characterize transcriptomes.It reveals precise gene structures and quantifies gene/transcript expressions [1][2][3][4][5] in various applications, such as variant calling [6], RNA editing analysis [7, 8], and gene fusion detection [9, 10].However, current widely used short read sequencing technologies have limited read length and systematic bias from library preparation. These drawbacks limit more accurate alignment [11] and precise gene isoform analysis [12], thus creating a bottleneck for transcriptomic studies.Two kinds of long read sequencing technologies, i.e., single molecule real time (SMRT) sequencing produced by Pacific Biosciences (PacBio) [13] and nanopore sequencing produced by Oxford Nanopore Technologies (ONT) [14], are emerging and promising to breakthrough the bottleneck of short reads in transcriptomic analysis. Both of them enable the production of much longer reads, the mean and maximum lengths of the reads being over ten to hundreds of thousands of base pairs (bp) [15,16], respectively. Taking this advantage, full-length transcripts can be sequenced by single reads, which is promising for substantially improving the accuracy of gene isoform reconstruction. Furthermore, there is less systematic bias in the sequencing procedure [17], which is also beneficial to gene/transcript expression quantification.Besides their advantages, PacBio and ONT reads have much higher sequencing error rates than that of short reads. For PacBio SMRT sequencing, the sequencing error rate of raw reads ("subreads")is about 10% to 20% [16]; for ONT nanopore sequencing, the sequencing error rates of 1D and 2D (also known as 1D 2 ) reads are about 25% and 12% [18,19], respectively. PacBio SMRT platforms can produce reads of inserts (ROIs) by sequencing circular fragments multiple times to largely reduce sequencing errors. However, this technology has lower sequencing yields and reduced read lengths. Therefore, these high sequencing errors raise new technical challenges for RNA-seq data analysis.Read alignment could be the most affected one, and the effect may not be limited to the read alignment itself since it is fundamental to many down...

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“…Circular Consensus Sequence (CCS) reads are a type of ROI and are generated by collapsing multiple subreads when ≥ 2 full-pass subreads are present. ONT produces longer reads with even higher error rates (error rates for "1D" raw reads: > 25%; error rates for "2D" consensus reads: 12-20%) (Križanović et al, 2018). Error-correction methods using short reads (such as the error correction tool LSC (Au et al, 2012)) have been created to correct the high rate of errors in long reads; however, error correction may create artifacts so that the corrected long reads may no longer be true single-molecule reads (Sharon et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Quantifying the Benefit Offered by Transcript Assembly on Single-Molecule Long Reads

Tung

Shao

Kingsford

2019

Preprint

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Third-generation sequencing technologies benefit transcriptome analysis by generating longer sequencing reads. However, not all single-molecule long reads represent full transcripts due to incomplete cDNA synthesis and the sequencing length limit of the platform. This drives a need for long read transcript assembly. We quantify the benefit that can be achieved by using a transcript assembler on long reads. Adding long-read-specific algorithms, we evolved Scallop to make Scallop-LR, a long-read transcript assembler, to handle the computational challenges arising from long read lengths and high error rates. Analyzing 26 SRA PacBio datasets using Scallop-LR, Iso-Seq Analysis, and StringTie, we quantified the amount by which assembly improved Iso-Seq results. Through combined evaluation methods, we found that Scallop-LR identifies 2100-4000 more (for 18 human datasets) or 1100-2200 more (for eight mouse datasets) known transcripts than Iso-Seq Analysis, which does not do assembly. Further, Scallop-LR finds 2.4-4.4 times more potentially novel isoforms than Iso-Seq Analysis for the human and mouse datasets. StringTie also identifies more transcripts than Iso-Seq Analysis. Adding long-read-specific optimizations in Scallop-LR increases the numbers of predicted known transcripts and potentially novel isoforms for the human transcriptome compared to several recent short-read assemblers (e.g. StringTie).Our findings indicate that transcript assembly by Scallop-LR can reveal a more complete human transcriptome.

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Evaluation of tools for long read RNA-seq splice-aware alignment

Abstract: Supplementary data are available at Bioinformatics online.

Cited by 67 publications

References 20 publications

Nanopore sequencing: Review of potential applications in functional genomics

Nanopore sequencing: Review of potential applications in functional genomics

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Quantifying the Benefit Offered by Transcript Assembly on Single-Molecule Long Reads

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