RIFRAF: a frame-resolving consensus algorithm

Eren, Kemal; Murrell, Ben

doi:10.1093/bioinformatics/bty426

Cited by 4 publications

(5 citation statements)

References 26 publications

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“…If the amplicon spans a coding sequence, then Rifraf.jl (12) can be used to infer a frame-shift corrected template sequence, as long as a reference sequence with a trusted reading frame is available.…”

Section: Methodsmentioning

confidence: 99%

“…(3) show that, for a 2.6 kb amplicon, under their quality filtering conditions, 80% of the errors are indels and 20% are substitution errors, and the indel errors are concentrated in homopolymer regions, increasing in rate with the length of the homopolymer. While high indel rates can be computationally challenging to deal with, since sequence alignment can be slow, they are favorable from a statistical perspective, because the errors appear in predictable places, making them more correctable (12).…”

Section: Introductionmentioning

confidence: 99%

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Long-read amplicon denoising

Kumar

Vollbrecht

Chernyshev

et al. 2019

Nucleic Acids Research

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Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called ‘amplicon denoising’, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Long-read amplicon denoising

Kumar

Vollbrecht

Chernyshev

et al. 2019

Nucleic Acids Research

Self Cite

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“…We exploit kmer seeding (k=30), and this approximate pairwise alignment algorithm scales linearly with sequence length. If the amplicon spans a coding sequence, then Rifraf.jl (10) can be used to infer a frame-shift corrected template sequence, as long as a reference sequence with a trusted reading frame is available.…”

Section: C3 Fine Cluster Splittingmentioning

confidence: 99%

“…(3) show that, for a 2.6kb amplicon, with a post-filtering error rate of 0.5%, 80% are indel and 20% substitution errors, and the indel errors are concentrated in homopolymer regions, increasing in rate with the length of the homopolymer. While high indel rates can be computationally challenging to deal with, since sequence alignment can be slow, they are favorable from a statistical perspective, because the errors appear in predictable places, making them more correctable (10). Amplicon denoising (11)(12)(13)(14)(15)(16)(17) refers to a process that takes a large set of reads, corrupted by sequencing errors, and attempts to distill the noiseless variants and their frequencies.…”

Section: Introductionmentioning

confidence: 99%

Long-read amplicon denoising

Kumar¹,

Vollbrecht²,

Chernyshev³

et al. 2018

Preprint

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† These authors contributed equally Long-read next generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies. Called "amplicon denoising", this problem has been extensively studied for short-read sequencing technologies, but current solutions do not appear to generalize well to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads (here ∼2.6kb) and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On one real dataset with ground truth, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method.Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD) are implemented purely in the Julia scientific computing language, and are hereby released along with a complete toolkit of functions that allow long-read amplicon sequence analysis pipelines to be constructed in pure Julia. Further, we make available a webserver to dramatically simplify the processing of long-read PacBio sequences.

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“…In future applications of CIDER-Seq where reference genomes are available (i.e. applications apart from conventional metagenomics), this low-level of frameshift error can be further corrected using existing long-read sequencing-specific frameshift-correction algorithms such as Frame-Pro (29) or RIFRAF (30).…”

Section: Resultsmentioning

confidence: 99%

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field

Mehta

Hirsch‐Hoffmann

Were

et al. 2018

Nucleic Acids Research

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We present a new method, CIDER-Seq (Circular DNA Enrichment sequencing) for the unbiased enrichment and long-read sequencing of viral-sized circular DNA molecules. We used CIDER-Seq to produce single-read full-length virus genomes for the first time. CIDER-Seq combines PCR-free virus enrichment with Single Molecule Real Time sequencing and a new sequence de-concatenation algorithm. We apply our technique to produce >1200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral double-stranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our method and its application in an economically important crop plant opens new possibilities in periodic virus sequence surveillance and accurate profiling of diverse circular DNA elements.

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RIFRAF: a frame-resolving consensus algorithm

Abstract: Supplementary data are available at Bioinformatics online.

Cited by 4 publications

References 26 publications

Long-read amplicon denoising

Long-read amplicon denoising

Long-read amplicon denoising

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field

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