SNP discovery using Next Generation Transcriptomic Sequencing in Atlantic herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Diagnostic value of increased sensitivity by massively parallel sequencing

Routine molecular diagnostics modalities are unable to confidently detect low frequency mutations (<5-15%) that may indicate response to targeted therapies. We confirm the presence of a low frequency NRAS mutation in a rectal cancer patient using massively parallel sequencing when previous Sanger sequencing results proved negative and Q-PCR testing inconclusive. There is increasing evidence that these low frequency mutations may confer resistance to anti-EGFR therapy. In view of negative/inconclusive Sanger sequencing and Q-PCR results for NRAS mutations in a KRAS wt rectal case, the diagnostic biopsy and 4 distinct subpopulations of cells in the resection specimen after conventional chemo/radiotherapy were massively parallel sequenced using the Ion Torrent PGM. DNA was derived from FFPE rectal cancer tissue and amplicons produced using the Cancer Hotspot Panel V2 and sequenced using semiconductor technology. NRAS mutations were observed at varying frequencies in the patient biopsy (12.2%) and all four subpopulations of cells in the resection with an average frequency of 7.3% (lowest 2.6%). The results of the NGS also provided the mutational status of 49 other genes that may have prognostic or predictive value, including KRAS and PIK3CA. NGS technology has been postulated in diagnostics because of its capability to generate results in large panels of clinically meaningful genes in a cost-effective manner. This case illustrates another potential advantage of this technology: its use for detecting low frequency mutations that may influence therapeutic decisions in cancer treatment.

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib.

METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies.

RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib.

CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

Novel Application of Genetic Sequencing algorithms to optimisation of hardware resource sharing for DSP

Field programmable gate array (FPGA) technology is a powerful platform for implementing computationally complex, digital signal processing (DSP) systems. Applications that are multi-modal, however, are designed for worse case conditions. In this paper, genetic sequencing techniques are applied to give a more sophisticated decomposition of the algorithmic variations, thus allowing an unified hardware architecture which gives a 10-25% area saving and 15% power saving for a digital radar receiver.

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding

This study examines the potential of next-generation sequencing based ‘genotyping-by-sequencing’ (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

LaGomiCs - Lagomorph Genomics Consortium: An International Collaborative Effort for Sequencing the Genomes of an Entire Mammalian Order

The order Lagomorpha comprises about 90 living species, divided in 2 families: the pikas (Family Ochotonidae), and the rabbits, hares, and jackrabbits (Family Leporidae). Lagomorphs are important economically and scientifically as major human food resources, valued game species, pests of agricultural significance, model laboratory animals, and key elements in food webs. A quarter of the lagomorph species are listed as threatened. They are native to all continents except Antarctica, and occur up to 5000 m above sea level, from the equator to the Arctic, spanning a wide range of environmental conditions. The order has notable taxonomic problems presenting significant difficulties for defining a species due to broad phenotypic variation, overlap of morphological characteristics, and relatively recent speciation events. At present, only the genomes of 2 species, the European rabbit (Oryctolagus cuniculus) and American pika (Ochotona princeps) have been sequenced and assembled. Starting from a paucity of genome information, the main scientific aim of the Lagomorph Genomics Consortium (LaGomiCs), born from a cooperative initiative of the European COST Action “A Collaborative European Network on Rabbit Genome Biology—RGB-Net” and the World Lagomorph Society (WLS), is to provide an international framework for the sequencing of the genome of all extant and selected extinct lagomorphs. Sequencing the genomes of an entire order will provide a large amount of information to address biological problems not only related to lagomorphs but also to all mammals. We present current and planned sequencing programs and outline the final objective of LaGomiCs possible through broad international collaboration.

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n 5 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Next-generation sequencing is highly sensitive for the detection of beta-catenin mutations in desmoid-type fibromatoses

Desmoid-type fibromatoses are locally aggressive and frequently recurrent tumours, and an accurate diagnosis is essential for patient management. The majority of sporadic lesions harbour beta-catenin (CTNNB1) mutations. We used next-generation sequencing to detect CTNNB1 mutations and to compare the sensitivity and specificity of next-generation sequencing with currently employed mutation detection techniques: mutation-specific restriction enzyme digestion and polymerase chain reaction amplification. DNA was extracted from formalin-fixed paraffin-embedded needle biopsy or resection tissue sections from 144 patients with sporadic desmoid-type fibromatoses, four patients with syndrome-related desmoid-type fibromatoses and 11 morphological mimics. Two primer pairs were designed for CTNNB1 mutation hotspots. Using ≥10 ng of DNA, libraries were generated by Fluidigm and sequenced on the Ion Torrent Personal Genome Machine. Next-generation sequencing had a sensitivity of 92.36 % (133/144, 95 % CIs: 86.74 to 96.12 %) and a specificity of 100 % for the detection of CTNNB1 mutations in desmoid-type fibromatoses-like spindle cell lesions. All mutations detected by mutation-specific restriction enzyme digestion were identified by next-generation sequencing. Next-generation sequencing identified additional mutations in 11 tumours that were not detected by mutation-specific restriction enzyme digestion, two of which have not been previously described. Next-generation sequencing is highly sensitive for the detection of CTNNB1 mutations. This multiplex assay has the advantage of detecting additional mutations compared to those detected by mutation-specific restriction enzyme digestion (sensitivity 82.41 %). The technology requires minimal DNA and is time- and cost-efficient.

Ultra-deep sequencing provides insights into the virology of hepatitis C super-infections in a case of three sequential infections with different genotypes

The current epidemic of Hepatitis C infection in HIV-positive men who have sex with men is associated with increasing use of recreational drugs. Multiple HCV infections have been reported in haemophiliacs and intravenous drug users. Using ultra-deep sequencing analysis, we present the case of an HIV-positive MSM with evidence of three sequential HCV infections, each occurring during the acute phase of the preceding infection, following risk exposures. We observed rapid replacement of the original strain by the incoming genotype at subsequent time points. The impact of HCV super-infection remains unclear and UDS may provide new insights.

Sequencing cutting patterns with colored interval graphs

The problem addressed here originates in the industry of flat glass cutting and wood panel sawing, where smaller items are cut from larger items accordingly to predefined cutting patterns. In this type of industry the smaller pieces that are cut from the patterns are piled around the machine in stacks according to the size of the pieces, which are moved to the warehouse only when all items of the same size have been cut. If the cutting machine can process only one pattern at a time, and the workspace is limited, it is desirable to set the sequence in which the cutting patterns are processed in a way to minimize the maximum number of open stacks around the machine. This problem is known in literature as the minimization of open stacks (MOSP). To find the best sequence of the cutting patterns, we propose an integer programming model, based on interval graphs, that searches for an appropriate edge completion of the given graph of the problem, while defining a suitable coloring of its vertices.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

Modeling sequencing errors by combining Hidden Markov models.

Among the largest resources for biological sequence data is the large amount of expressed sequence tags (ESTs) available in public and proprietary databases. ESTs provide information on transcripts but for technical reasons they often contain sequencing errors. Therefore, when analyzing EST sequences computationally, such errors must be taken into account. Earlier attempts to model error prone coding regions have shown good performance in detecting and predicting these while correcting sequencing errors using codon usage frequencies. In the research presented here, we improve the detection of translation start and stop sites by integrating a more complex mRNA model with codon usage bias based error correction into one hidden Markov model (HMM), thus generalizing this error correction approach to more complex HMMs. We show that our method maintains the performance in detecting coding sequences.

An atlas of human gene expression from massively parallel signature sequencing (MPSS).

We have used massively parallel signature sequencing (MPSS) to sample the transcriptomes of 32 normal human tissues to an unprecedented depth, thus documenting the patterns of expression of almost 20,000 genes with high sensitivity and specificity. The data confirm the widely held belief that differences in gene expression between cell and tissue types are largely determined by transcripts derived from a limited number of tissue-specific genes, rather than by combinations of more promiscuously expressed genes. Expression of a little more than half of all known human genes seems to account for both the common requirements and the specific functions of the tissues sampled. A classification of tissues based on patterns of gene expression largely reproduces classifications based on anatomical and biochemical properties. The unbiased sampling of the human transcriptome achieved by MPSS supports the idea that most human genes have been mapped, if not functionally characterized. This data set should prove useful for the identification of tissue-specific genes, for the study of global changes induced by pathological conditions, and for the definition of a minimal set of genes necessary for basic cell maintenance. The data are available on the Web at http://mpss.licr.org and http://sgb.lynxgen.com.