The clonal and mutational evolution spectrum of primary triple-negative breast cancers.

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status.

Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional "noise," due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer.

Conserved role of intragenic DNA methylation in regulating alternative promoters.

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution.

Recent advances in next generation sequencing have made it possible to precisely characterize all somatic coding mutations that occur during the development and progression of individual cancers. Here we used these approaches to sequence the genomes (>43-fold coverage) and transcriptomes of an oestrogen-receptor-alpha-positive metastatic lobular breast cancer at depth. We found 32 somatic non-synonymous coding mutations present in the metastasis, and measured the frequency of these somatic mutations in DNA from the primary tumour of the same patient, which arose 9 years earlier. Five of the 32 mutations (in ABCB11, HAUS3, SLC24A4, SNX4 and PALB2) were prevalent in the DNA of the primary tumour removed at diagnosis 9 years earlier, six (in KIF1C, USP28, MYH8, MORC1, KIAA1468 and RNASEH2A) were present at lower frequencies (1-13%), 19 were not detected in the primary tumour, and two were undetermined. The combined analysis of genome and transcriptome data revealed two new RNA-editing events that recode the amino acid sequence of SRP9 and COG3. Taken together, our data show that single nucleotide mutational heterogeneity can be a property of low or intermediate grade primary breast cancers and that significant evolution can occur with disease progression.

HealthPathways: creating a pathway for health systems reform.

Inefficiencies in the co-ordination and integration of primary and secondary care services in Australia, have led to increases in waiting times, unnecessary presentations to emergency departments and issues around poor discharge of patients. HealthPathways is a program developed in Canterbury, New Zealand, that builds relationships between General Practitioners and Specialists and uses information technology so that efficiency is maximised and the right patient is given the right care at the right time. Healthpathways is being implemented by a number of Medicare Locals across Australia however, little is known about the impact HealthPathways may have in Australia. This article provides a short description of HealthPathways and considers what it may offer in the Australian context and some of the barriers and facilitators to implementation.

Whole-genome sequencing and social-network analysis of a tuberculosis outbreak.

BACKGROUND: An outbreak of tuberculosis occurred over a 3-year period in a medium-size community in British Columbia, Canada. The results of mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) genotyping suggested the outbreak was clonal. Traditional contact tracing did not identify a source. We used whole-genome sequencing and social-network analysis in an effort to describe the outbreak dynamics at a higher resolution. METHODS: We sequenced the complete genomes of 32 Mycobacterium tuberculosis outbreak isolates and 4 historical isolates (from the same region but sampled before the outbreak) with matching genotypes, using short-read sequencing. Epidemiologic and genomic data were overlaid on a social network constructed by means of interviews with patients to determine the origins and transmission dynamics of the outbreak. RESULTS: Whole-genome data revealed two genetically distinct lineages of M. tuberculosis with identical MIRU-VNTR genotypes, suggesting two concomitant outbreaks. Integration of social-network and phylogenetic analyses revealed several transmission events, including those involving "superspreaders." Both lineages descended from a common ancestor and had been detected in the community before the outbreak, suggesting a social, rather than genetic, trigger. Further epidemiologic investigation revealed that the onset of the outbreak coincided with a recorded increase in crack cocaine use in the community. CONCLUSIONS: Through integration of large-scale bacterial whole-genome sequencing and social-network analysis, we show that a socioenvironmental factor--most likely increased crack cocaine use--triggered the simultaneous expansion of two extant lineages of M. tuberculosis that was sustained by key members of a high-risk social network. Genotyping and contact tracing alone did not capture the true dynamics of the outbreak. (Funded by Genome British Columbia and others.).

Complete genomic landscape of a recurring sporadic parathyroid carcinoma.

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto-oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high-throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well-characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets.

Enhancing the implementation of provider-to-provider telehealth in rural and remote areas: A mixed methods study protocol.

Background: Virtual healthcare solutions are proposed as a way to combat the inequity of access to healthcare in rural and remote areas, and to better support the front-line providers who work in these areas. Rural provider-to-provider telehealth (RPPT) connects rural and remote clinicians to a 'hub' of healthcare specialists who can increase access to emergency and specialised healthcare via an integrated model. Reported benefits for the place-based provider include enhanced knowledge, expanded professional development opportunities, improved scope of practice, and increased confidence in treating more complex cases. These reported benefits could have implications for supporting and futureproofing our health workforce in terms of productivity, burnout, recruitment, and retention. Methods: The research uses an explanatory sequential mixed methods approach across multiple phases to evaluate the current implementation of Western Australia Country Health Service's (WACHS) Command Centre (CC) services and explore factors associated with their differential use. The primary population of interest and participants in this study are the place-based providers in country Western Australia (WA). Patient data constitutes the secondary population, informing the access and reach of CC services into country WA. Data collection will include service data, an online survey, and semi-structured interviews with the primary population. The data will be interpreted to inform evidence-based strategies and recommendations to improve the implementation and sustainment of RPPT. Discussion: Innovative and sustained workforce models and solutions are needed globally. Virtual healthcare, including provider-to-provider models, demonstrate potential, especially in rural and remote areas, designed to increase access to specialised expertise for patients and to support the local workforce. This research will generate new data around behaviour, perceptions, and value from the WACHS rural and remote workforce about provider-to-provider telehealth, to explore the implementation and investigate strategies for the long-term sustainment of RPPT services.

Barnacle: detecting and characterizing tandem duplications and fusions in transcriptome assemblies.

BACKGROUND: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers. RESULTS: We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets. CONCLUSIONS: Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.

De novo assembly and analysis of RNA-seq data.

We describe Trans-ABySS, a de novo short-read transcriptome assembly and analysis pipeline that addresses variation in local read densities by assembling read substrings with varying stringencies and then merging the resulting contigs before analysis. Analyzing 7.4 gigabases of 50-base-pair paired-end Illumina reads from an adult mouse liver poly(A) RNA library, we identified known, new and alternative structures in expressed transcripts, and achieved high sensitivity and specificity relative to reference-based assembly methods.

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers.

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.

Public preference on sharing health data to inform research, health policy and clinical practice in Australia: A stated preference experiment.

OBJECTIVE: To investigate public willingness to share sensitive health information for research, health policy and clinical practice. METHODS: A total of 1,003 Australian respondents answered an online, attribute-driven, survey in which participants were asked to accept or reject hypothetical choice sets based on a willingness to share their health data for research and frontline-medical support as part of an integrated health system. The survey consisted of 5 attributes: Stakeholder access for analysis (Analysing group); Type of information collected; Purpose of data collection; Information governance; and Anticipated benefit; the results of which were analysed using logistic regression. RESULTS: When asked about their preference for sharing their health data, respondents had no preference between data collection for the purposes of clinical practice, health policy or research, with a slight preference for having government organisations manage, govern and curate the integrated datasets from which the analysis was being conducted. The least preferred option was for personal health records to be integrated with insurance records or for their data collected by privately owned corporate organisations. Individuals preferred their data to be analysed by a public healthcare provider or government staff and expressed a dislike for any private company involvement. CONCLUSIONS: The findings from this study suggest that Australian consumers prefer to share their health data when there is government oversight, and have concerns about sharing their anonymised health data for clinical practice, health policy or research purposes unless clarity is provided pertaining to its intended purpose, limitations of use and restrictions to access. Similar findings have been observed in the limited set of existing international studies utilising a stated preference approach. Evident from this study, and supported by national and international research, is that the establishment and preservation of a social license for data linkage in health research will require routine public engagement as a result of continuously evolving technological advancements and fluctuating risk tolerance. Without more work to understand and address stakeholder concerns, consumers risk being reluctant to participate in data-sharing and linkage programmes.

Mutation of FOXL2 in granulosa-cell tumors of the ovary.

BACKGROUND: Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS: We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS: All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS: Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Australian general practitioner perceptions to sharing clinical data for secondary use: a mixed method approach.

OBJECTIVE: The potential for data collected in general practice to be linked and used to address health system challenges of maintaining quality care, accessibility and safety, including pandemic support, has led to an increased interest in public acceptability of data sharing, however practitioners have rarely been asked to share their opinions on the topic. This paper attempts to gain an understanding of general practitioner's perceptions on sharing routinely collected data for the purposes of healthcare planning and research. It also compares findings with data sharing perceptions in an international context.  MATERIALS AND METHODS: A mixed methods approach combining an initial online survey followed by face-to-face interviews (before and during COVID-19), designed to identify the barriers and facilitators to sharing data, were conducted on a cross sectional convenience sample of general practitioners across Western Australia (WA). RESULTS: Eighty online surveys and ten face-to-face interviews with general practitioners were conducted from November 2020 - May 2021. Although respondents overwhelmingly identified the importance of population health research, their willingness to participate in data sharing programs was determined by a perception of trust associated with the organisation collecting and analysing shared data; a clearly defined purpose and process of collected data; including a governance structure providing confidence in the data sharing initiative simultaneously enabling a process of data sovereignty and autonomy. DISCUSSION: Results indicate strong agreement around the importance of sharing patient's medical data for population and health research and planning. Concerns pertaining to lack of trust, governance and secondary use of data continue to be a setback to data sharing with implications for primary care business models being raised. CONCLUSION: To further increase general practitioner's confidence in sharing their clinical data, efforts should be directed towards implementing a robust data governance structure with an emphasis on transparency and representative stakeholder inclusion as well as identifying the role of government and government funded organisations, as well as building trust with the entities collecting and analysing the data.

De novo transcriptome assembly with ABySS.

MOTIVATION: Whole transcriptome shotgun sequencing data from non-normalized samples offer unique opportunities to study the metabolic states of organisms. One can deduce gene expression levels using sequence coverage as a surrogate, identify coding changes or discover novel isoforms or transcripts. Especially for discovery of novel events, de novo assembly of transcriptomes is desirable. RESULTS: Transcriptome from tumor tissue of a patient with follicular lymphoma was sequenced with 36 base pair (bp) single- and paired-end reads on the Illumina Genome Analyzer II platform. We assembled approximately 194 million reads using ABySS into 66 921 contigs 100 bp or longer, with a maximum contig length of 10 951 bp, representing over 30 million base pairs of unique transcriptome sequence, or roughly 1% of the genome. AVAILABILITY AND IMPLEMENTATION: Source code and binaries of ABySS are freely available for download at http://www.bcgsc.ca/platform/bioinfo/software/abyss. Assembler tool is implemented in C++. The parallel version uses Open MPI. ABySS-Explorer tool is implemented in Java using the Java universal network/graph framework. CONTACT: ibirol@bcgsc.ca.

Global analysis of in vivo Foxa2-binding sites in mouse adult liver using massively parallel sequencing.

Foxa2 (HNF3 beta) is a one of three, closely related transcription factors that are critical to the development and function of the mouse liver. We have used chromatin immunoprecipitation and massively parallel Illumina 1G sequencing (ChIP-Seq) to create a genome-wide profile of in vivo Foxa2-binding sites in the adult liver. More than 65% of the approximately 11.5 k genomic sites associated with Foxa2 binding, mapped to extended gene regions of annotated genes, while more than 30% of intragenic sites were located within first introns. 20.5% of all sites were further than 50 kb from any annotated gene, suggesting an association with novel gene regions. QPCR analysis demonstrated a strong positive correlation between peak height and fold enrichment for Foxa2-binding sites. We measured the relationship between Foxa2 and liver gene expression by overlapping Foxa2-binding sites with a SAGE transcriptome profile, and found that 43.5% of genes expressed in the liver were also associated with Foxa2 binding. We also identified potential Foxa2-interacting transcription factors whose motifs were enriched near Foxa2-binding sites. Our comprehensive results for in vivo Foxa2-binding sites in the mouse liver will contribute to resolving transcriptional regulatory networks that are important for adult liver function.

Evaluation of HealthPathways: an appraisal of usage, experiences and opinions of healthcare professionals in Australia and New Zealand.

Objectives HealthPathways, pioneered in Canterbury, New Zealand, in 2008, is a web-based tool designed to promote health care integration and patient management in primary care and to reduce fragmentation in the delivery of health services. This cross-sectional study evaluated the utilisation and perceptions of this tool among health professionals in Australia and New Zealand. Methods A cross-sectional survey was administered online through Research Electronic Data Capture (REDCap) to general practitioners (GPs), practice nurses and managers, nurse practitioners, specialist and community nurses, hospital clinicians, nurses, managers, and allied health professionals between April and September 2018. The frequency of HealthPathways use in the previous month was modelled as an ordered response using an ordered logistic regression model after adjusting for the possible effects of sex, age, years in clinical practice, location and time spent in practice. Results Health professionals perceived HealthPathways to be useful in primary care management and referral, as well as in the prereferral treatment of patients. GPs in New Zealand, New South Wales and Victoria were 73%, 47% and 27% more likely to have used HealthPathways ≥10 times in the previous month respectively. Conclusion The results suggest that HealthPathways is having a positive effect on healthcare systems in New Zealand and Australia. However, differences in uptake suggests the need for focused implementation, integration into eReferral software and expanding the tool to medical students, registrars, allied health professionals and potentially patients to encourage behavioural change. What is known about the topic? Early evaluations suggest that HealthPathways is a useful tool for health professionals, although uptake and utilisation may be limited. However, there is no comparative evidence regarding uptake and implementation of the tool. What does the paper add? This study is among the first to provide a comparative narrative of the literature assessing the implementation and uptake of HealthPathways across Australia and New Zealand. It is also among the first to compare the perceptions of allied health professionals in the use of HealthPathways across Australia and New Zealand. What are the implications for practitioners? The results of this study suggest the need for focused implementation, integration into eReferral software and expanding the tool to medical students, registrars, allied health professionals and potentially patients to encourage behavioural change.

FindPeaks 3.1: a tool for identifying areas of enrichment from massively parallel short-read sequencing technology.

SUMMARY: Next-generation sequencing can provide insight into protein-DNA association events on a genome-wide scale, and is being applied in an increasing number of applications in genomics and meta-genomics research. However, few software applications are available for interpreting these experiments. We present here an efficient application for use with chromatin-immunoprecipitation (ChIP-Seq) experimental data that includes novel functionality for identifying areas of gene enrichment and transcription factor binding site locations, as well as for estimating DNA fragment size distributions in enriched areas. The FindPeaks application can generate UCSC compatible custom 'WIG' track files from aligned-read files for short-read sequencing technology. The software application can be executed on any platform capable of running a Java Runtime Environment. Memory requirements are proportional to the number of sequencing reads analyzed; typically 4 GB permits processing of up to 40 million reads. AVAILABILITY: The FindPeaks 3.1 package and manual, containing algorithm descriptions, usage instructions and examples, are available at http://www.bcgsc.ca/platform/bioinfo/software/findpeaks Source files for FindPeaks 3.1 are available for academic use.

Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing.

Sequence-based methods for transcriptome characterization have typically relied on generation of either serial analysis of gene expression tags or expressed sequence tags. Although such approaches have the potential to enumerate transcripts by counting sequence tags derived from them, they typically do not robustly survey the majority of transcripts along their entire length. Here we show that massively parallel sequencing of randomly primed cDNAs, using a next-generation sequencing-by-synthesis technology, offers the potential to generate relative measures of mRNA and individual exon abundance while simultaneously profiling the prevalence of both annotated and novel exons and exon-splicing events. This technique identifies known single nucleotide polymorphisms (SNPs) as well as novel single-base variants. Analysis of these variants, and previously unannotated splicing events in the HeLa S3 cell line, reveals an overrepresentation of gene categories including those previously implicated in cancer.