Assembling genomes using short-read sequencing technology

Short-read sequencing technology can bring gigabase genome assemblies in under a million dollars

Sealer: a scalable gap-closing application for finishing draft genomes

BACKGROUND: While next-generation sequencing technologies have made sequencing genomes faster and more affordable, deciphering the complete genome sequence of an organism remains a significant bioinformatics challenge, especially for large genomes. Low sequence coverage, repetitive elements and short read length make de novo genome assembly difficult, often resulting in sequence and/or fragment “gaps” – uncharacterized nucleotide (N) stretches of unknown or estimated lengths. Some of these gaps can be closed by re-processing latent information in the raw reads. Even though there are several tools for closing gaps, they do not easily scale up to processing billion base pair genomes. RESULTS: Here we describe Sealer, a tool designed to close gaps within assembly scaffolds by navigating de Bruijn graphs represented by space-efficient Bloom filter data structures. We demonstrate how it scales to successfully close 50.8 % and 13.8 % of gaps in human (3 Gbp) and white spruce (20 Gbp) draft assemblies in under 30 and 27 h, respectively – a feat that is not possible with other leading tools with the breadth of data used in our study. CONCLUSION: Sealer is an automated finishing application that uses the succinct Bloom filter representation of a de Bruijn graph to close gaps in draft assemblies, including that of very large genomes. We expect Sealer to have broad utility for finishing genomes across the tree of life, from bacterial genomes to large plant genomes and beyond. Sealer is available for download at https://github.com/bcgsc/abyss/tree/sealer-release. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0663-4) contains supplementary material, which is available to authorized users

The genetic landscape of high-risk neuroblastoma

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%1. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 cases using a combination of whole exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per megabase (0.48 non-silent), and remarkably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, an additional 7.1% had focal deletions), MYCN (1.7%, a recurrent p.Pro44Leu alteration), and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1, and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies reliant upon frequently altered oncogenic drivers

Spaced Seed Data Structures for De Novo

De novo assembly of the genome of a species is essential in the absence of a reference genome sequence. Many scalable assembly algorithms use the de Bruijn graph (DBG) paradigm to reconstruct genomes, where a table of subsequences of a certain length is derived from the reads, and their overlaps are analyzed to assemble sequences. Despite longer subsequences unlocking longer genomic features for assembly, associated increase in compute resources limits the practicability of DBG over other assembly archetypes already designed for longer reads. Here, we revisit the DBG paradigm to adapt it to the changing sequencing technology landscape and introduce three data structure designs for spaced seeds in the form of paired subsequences. These data structures address memory and run time constraints imposed by longer reads. We observe that when a fixed distance separates seed pairs, it provides increased sequence specificity with increased gap length. Further, we note that Bloom filters would be suitable to implicitly store spaced seeds and be tolerant to sequencing errors. Building on this concept, we describe a data structure for tracking the frequencies of observed spaced seeds. These data structure designs will have applications in genome, transcriptome and metagenome assemblies, and read error correction

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest

Background The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. Results We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. Conclusions Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species

Background: The process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly. Results: In Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies. Conclusions: Many current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society