Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools

Background

Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality.

Results

In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS.

Conclusions

By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-439) contains supplementary material, which is available to authorized users.     

Distinguishing between driver and passenger mutations in individual cancer genomes by network enrichment analysis

Background

In somatic cancer genomes, delineating genuine driver mutations against a background of multiple passenger events is a challenging task. The difficulty of determining function from sequence data and the low frequency of mutations are increasingly hindering the search for novel, less common cancer drivers. The accumulation of extensive amounts of data on somatic point and copy number alterations necessitates the development of systematic methods for driver mutation analysis.

Results

We introduce a framework for detecting driver mutations via functional network analysis, which is applied to individual genomes and does not require pooling multiple samples. It probabilistically evaluates 1) functional network links between different mutations in the same genome and 2) links between individual mutations and known cancer pathways. In addition, it can employ correlations of mutation patterns in pairs of genes. The method was used to analyze genomic alterations in two TCGA datasets, one for glioblastoma multiforme and another for ovarian carcinoma, which were generated using different approaches to mutation profiling. The proportions of drivers among the reported de novo point mutations in these cancers were estimated to be 57.8% and 16.8%, respectively. The both sets also included extended chromosomal regions with synchronous duplications or losses of multiple genes. We identified putative copy number driver events within many such segments. Finally, we summarized seemingly disparate mutations and discovered a functional network of collagen modifications in the glioblastoma. In order to select the most efficient network for use with this method, we used a novel, ROC curve-based procedure for benchmarking different network versions by their ability to recover pathway membership.

Conclusions

The results of our network-based procedure were in good agreement with published gold standard sets of cancer genes and were shown to complement and expand frequency-based driver analyses. On the other hand, three sequence-based methods applied to the same data yielded poor agreement with each other and with our results. We review the difference in driver proportions discovered by different sequencing approaches and discuss the functional roles of novel driver mutations. The software used in this work and the global network of functional couplings are publicly available at http://research.scilifelab.se/andrej_alexeyenko/downloads.html.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-308) contains supplementary material, which is available to authorized users.     

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

Subtype Classification of Iranian HIV-1 Sequences Registered in the HIV Databases, 2006-2013

Background

The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the past few years. While the earliest cases were among hemophiliacs, injection drug users (IDUs) fuel the current epidemic. Previous molecular epidemiological analysis found that subtype A was most common among IDUs but more recent studies suggest CRF_35AD may be more prevalent now. To gain a better understanding of the molecular epidemiology of HIV-1 infection in Iran, we analyzed all Iranian HIV sequence data from the Los Alamos National Laboratory.

Methods

All Iranian HIV sequences from subtyping studies with pol, gag, env and full-length HIV-1 genome sequences registered in the HIV databases (www.hiv.lanl.gov) between 2006 and 2013 were downloaded. Phylogenetic trees of each region were constructed using Neighbor-Joining (NJ) and Maximum Parsimony methods.

Results

A total of 475 HIV sequences were analyzed. Overall, 78% of sequences were CRF_35AD. By gene region, CRF_35AD comprised 83% of HIV-1 pol, 62% of env, 78% of gag, and 90% of full-length genome sequences analyzed. There were 240 sequences re-categorized as CRF_AD. The proportion of CRF_35AD sequences categorized by the present study is nearly double the proportion of what had been reported.

Conclusions

Phylogenetic analysis indicates HIV-1 subtype CRF_35AD is the predominant circulating strain in Iran. This result differed from previous studies that reported subtype A as most prevalent in HIV- infected patients but confirmed other studies which reported CRF_35AD as predominant among IDUs. The observed epidemiological connection between HIV strains circulating in Iran and Afghanistan may be due to drug trafficking and/or immigration between the two countries. This finding suggests the possible origins and transmission dynamics of HIV/AIDS within Iran and provides useful information for designing control and intervention strategies.     

IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

Motivation

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads.

Results

We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity.

Availability and Implementation

IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.     

Oral Doxycycline Reduces Pterygium Lesions; Results from a Double Blind,Randomized, Placebo Controlled Clinical Trial

Purpose

To determine whether oral doxycycline treatment reduces pterygium lesions.

Design

Double blind, randomized, placebo controlled clinical trial.

Participants

98 adult patients with primary pterygium.

Methods

Patients were randomly assigned to receive 100 mg oral doxycycline twice a day (49 subjects), or placebo (49 subjects), for 30 days. Photographs of the lesion were taken at the time of recruitment and at the end of the treatment. Follow-up sessions were performed 6 and 12 months post-treatment. Statistical analyses for both continuous and categorical variables were applied. p values of less than 0.05 were considered to indicate statistical significance.

Main Outcome Measures

The primary endpoint was the change in lesion size after 30 days of treatment.

Results

The primary endpoint was not met for the whole population but subgroup analysis showed that doxycycline was effective in patients of Caucasian origin while other ethnicities, mostly Hispanic, did not respond to the treatment. Moreover, there was a correlation between age and better response (p = 0.003). Adverse events were uncommon, mild, and in agreement with previous reports on short doxycycline treatments.

Conclusions

Oral doxycycline was superior to placebo for the treatment of primary pterygia in older Caucasian patients. These findings support the use of doxycycline for pterygium treatment in particular populations.

Trial Registration

European Union Clinical Trials Register EudraCT 2008-007178-39     

Synthesis of 53 tissue and cell line expression QTL datasets reveals master eQTLs

Background

Gene expression genetic studies in human tissues and cells identify cis- and trans-acting expression quantitative trait loci (eQTLs). These eQTLs provide insights into regulatory mechanisms underlying disease risk. However, few studies systematically characterized eQTL results across cell and tissues types. We synthesized eQTL results from >50 datasets, including new primary data from human brain, peripheral plaque and kidney samples, in order to discover features of human eQTLs.

Results

We find a substantial number of robust cis-eQTLs and far fewer trans-eQTLs consistent across tissues. Analysis of 45 full human GWAS scans indicates eQTLs are enriched overall, and above nSNPs, among positive statistical signals in genetic mapping studies, and account for a significant fraction of the strongest human trait effects. Expression QTLs are enriched for gene centricity, higher population allele frequencies, in housekeeping genes, and for coincidence with regulatory features, though there is little evidence of 5′ or 3′ positional bias. Several regulatory categories are not enriched including microRNAs and their predicted binding sites and long, intergenic non-coding RNAs. Among the most tissue-ubiquitous cis-eQTLs, there is enrichment for genes involved in xenobiotic metabolism and mitochondrial function, suggesting these eQTLs may have adaptive origins. Several strong eQTLs (CDK5RAP2, NBPFs) coincide with regions of reported human lineage selection. The intersection of new kidney and plaque eQTLs with related GWAS suggest possible gene prioritization. For example, butyrophilins are now linked to arterial pathogenesis via multiple genetic and expression studies. Expression QTL and GWAS results are made available as a community resource through the NHLBI GRASP database [http://apps.nhlbi.nih.gov/grasp/].

Conclusions

Expression QTLs inform the interpretation of human trait variability, and may account for a greater fraction of phenotypic variability than protein-coding variants. The synthesis of available tissue eQTL data highlights many strong cis-eQTLs that may have important biologic roles and could serve as positive controls in future studies. Our results indicate some strong tissue-ubiquitous eQTLs may have adaptive origins in humans. Efforts to expand the genetic, splicing and tissue coverage of known eQTLs will provide further insights into human gene regulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-532) contains supplementary material, which is available to authorized users.     

SHEAR: sample heterogeneity estimation and assembly by reference

Background

Personal genome assembly is a critical process when studying tumor genomes and other highly divergent sequences. The accuracy of downstream analyses, such as RNA-seq and ChIP-seq, can be greatly enhanced by using personal genomic sequences rather than standard references. Unfortunately, reads sequenced from these types of samples often have a heterogeneous mix of various subpopulations with different variants, making assembly extremely difficult using existing assembly tools. To address these challenges, we developed SHEAR (Sample Heterogeneity Estimation and Assembly by Reference; http://vk.cs.umn.edu/SHEAR), a tool that predicts SVs, accounts for heterogeneous variants by estimating their representative percentages, and generates personal genomic sequences to be used for downstream analysis.

Results

By making use of structural variant detection algorithms, SHEAR offers improved performance in the form of a stronger ability to handle difficult structural variant types and better computational efficiency. We compare against the lead competing approach using a variety of simulated scenarios as well as real tumor cell line data with known heterogeneous variants. SHEAR is shown to successfully estimate heterogeneity percentages in both cases, and demonstrates an improved efficiency and better ability to handle tandem duplications.

Conclusion

SHEAR allows for accurate and efficient SV detection and personal genomic sequence generation. It is also able to account for heterogeneous sequencing samples, such as from tumor tissue, by estimating the subpopulation percentage for each heterogeneous variant.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-84) contains supplementary material, which is available to authorized users.     

AdmixKJump: identifying population structure in recently diverged groups

Motivation

Correctly modeling population structure is important for understanding recent evolution and for association studies in humans. While pre-existing knowledge of population history can be used to specify expected levels of subdivision, objective metrics to detect population structure are important and may even be preferable for identifying groups in some situations. One such metric for genomic scale data is implemented in the cross-validation procedure of the program ADMIXTURE, but it has not been evaluated on recently diverged and potentially cryptic levels of population structure. Here, I develop a new method, AdmixKJump, and test both metrics under this scenario.

Findings

I show that AdmixKJump is more sensitive to recent population divisions compared to the cross-validation metric using both realistic simulations, as well as 1000 Genomes Project European genomic data. With two populations of 50 individuals each, AdmixKJump is able to detect two populations with 100% accuracy that split at least 10KYA, whereas cross-validation obtains this 100% level at 14KYA. I also show that AdmixKJump is more accurate with fewer samples per population. Furthermore, in contrast to the cross-validation approach, AdmixKJump is able to detect the population split between the Finnish and Tuscan populations of the 1000 Genomes Project.

Conclusion

AdmixKJump has more power to detect the number of populations in a cohort of samples with smaller sample sizes and shorter divergence times.

Availability

A java implementation can be found at https://sites.google.com/site/igsevolgenomicslab/home/downloads     

Optimising Controlled Human Malaria Infection Studies Using Cryopreserved P. falciparum Parasites Administered by Needle and Syringe

Background

Controlled human malaria infection (CHMI) studies have become a routine tool to evaluate efficacy of candidate anti-malarial drugs and vaccines. To date, CHMI trials have mostly been conducted using the bite of infected mosquitoes, restricting the number of trial sites that can perform CHMI studies. Aseptic, cryopreserved P. falciparum sporozoites (PfSPZ Challenge) provide a potentially more accurate, reproducible and practical alternative, allowing a known number of sporozoites to be administered simply by injection.

Methodology

We sought to assess the infectivity of PfSPZ Challenge administered in different dosing regimens to malaria-naive healthy adults (n = 18). Six participants received 2,500 sporozoites intradermally (ID), six received 2,500 sporozoites intramuscularly (IM) and six received 25,000 sporozoites IM.

Findings

Five out of six participants receiving 2,500 sporozoites ID, 3/6 participants receiving 2,500 sporozoites IM and 6/6 participants receiving 25,000 sporozoites IM were successfully infected. The median time to diagnosis was 13.2, 17.8 and 12.7 days for 2,500 sporozoites ID, 2,500 sporozoites IM and 25,000 sporozoites IM respectively (Kaplan Meier method; p = 0.024 log rank test).

Conclusions

2,500 sporozoites ID and 25,000 sporozoites IM have similar infectivities. Given the dose response in infectivity seen with IM administration, further work should evaluate increasing doses of PfSPZ Challenge IM to identify a dosing regimen that reliably infects 100% of participants.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01465048","term_id":"NCT01465048"}}NCT01465048