Near-optimal assembly for shotgun sequencing with noisy reads

Lam, Ka-Kit; Khalak, Asif; Tse, David

doi:10.1186/1471-2105-15-s9-s4

Cited by 46 publications

(28 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With current sequencing chemistries, PacBio read lengths are ∼16 kb on average but reach ∼50 kb, which can bridge repetitive regions not easily resolved with short read technology. Although PacBio reads have a high error rate (∼15%), because these errors are randomly distributed, several approaches can correct the reads for use in de novo assembly (Koren et al 2012;Ross et al 2013;Chaisson et al 2014;Lam et al 2014). Hybrid approaches use deep coverage from Illumina reads for error correction of the raw PacBio reads (Koren et al 2012).…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster

2017

View full text Add to dashboard Cite

Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. Here, we use single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. We determine the optimal de novo assembly methods and parameter combinations required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. We determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. We find evidence for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin.

show abstract

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster

2017

View full text Add to dashboard Cite

show abstract

“…Theoretical analyses of assembly show that error-prone reads are nearly as informative as errorfree reads, suggesting that read accuracy is less important than length 43,44 . Unicycler's performance on the simulated read sets matched these findings.…”

Section: Hybrid Assembly Of Simulated Long and Short Read Datasetsmentioning

confidence: 99%

Unicycler: resolving bacterial genome assemblies from short and long sequencing reads

Wick

Judd

Gorrie

et al. 2016

Preprint

137

147

View full text Add to dashboard Cite

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and errorprone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate "hybrid" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

show abstract

“…Paired end short reads from different sized longer inserts can improve contiguity, but uncertainty of fragment length and the lack of sequence between the insert ends makes resolving many repetitive structures challenging (5). Longer reads can circumvent this problem, even when such reads exhibit errors rates as high as 20% (5–8). Importantly, error-prone reads can be corrected, provided there is sufficient coverage and the errors are approximately uniformly distributed.…”

Section: Introductionmentioning

confidence: 99%

“…Despite these successes, shepherding a genome project through the process of DNA isolation, sequencing and assembly is still a challenge, especially for research groups for whom genomes are a means to another goal rather than the goal itself. For example, because high quality genome assembly relies upon long sequencing reads to bridge repetitive genomic regions (6,8,16,17) and high coverage to circumvent read errors (4,7,12), the stringent DNA isolation requirements (size, quantity and purity) for PacBio sequencing (10) intended for genome assembly are higher than those typically employed. Moreover, at present, the low average read quality produced by PacBio sequencing causes coverage requirements to be at least 50-fold (5,13,15).…”

Section: Introductionmentioning

confidence: 99%

Contiguous and accuratede novoassembly of metazoan genomes with modest long read coverage

Chakraborty¹,

Baldwin-Brown²,

Long³

et al. 2016

Nucleic Acids Res

352

294

View full text Add to dashboard Cite

Genome assemblies that are accurate, complete and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a ‘missing manual’ that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.

show abstract

Near-optimal assembly for shotgun sequencing with noisy reads

Cited by 46 publications

References 18 publications

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster

Unicycler: resolving bacterial genome assemblies from short and long sequencing reads

Contiguous and accuratede novoassembly of metazoan genomes with modest long read coverage

Contact Info

Product

Resources

About