Paired-end Sequencing Reads Research Articles

CBFβ/MYH11 Hypomethylation Signature In Patients With Acute Myeloid Leukaemia

It has been shown that changes in DNA methylation pattern in patients with acute myeloid leukaemia (AML) may both reflect specific molecular abnormalities or characterize a group of patients without a known molecular aberration. Studying genes that are epigenetically deregulated in different groups of patients may contribute to a more detailed understanding of pathways involved in leukemic transformation.For 15 samples (14 AMLs of diverse clinical and genetic background and pool of CD34+ control samples) SureSelectXT Human Methyl-Seq system (Agilent) was used to interrogate DNA methylation of selected regions (84 megabases in total) of their genomes. Targeted bisulfite libraries were run on a HiSeqTM2000 sequencer (Illumina) using 105 bp paired-end sequencing reads. Methylation status of candidate regions was verified using 454 bisulfite pyrosequencing (Roche). The expression levels of selected genes were assessed with TaqMan Gene Expression Assays (Applied Biosystems).Targeted methylation data was correlated with previously obtained whole-genome bisulfite (WGBS) data for the cross-experiment (WGBS/target enrichment) samples (n=3), taking into account positions with minimal coverage of 10. There was a strong positive correlation (R≥0.97) for all three samples. Pairwise Pearson correlations were computed for all (targeted and WGBS) samples and formed the basis of a hierarchical clustering analysis. The cross-experiment replicates clustered together. From the clinical and molecular characteristics, only CBFβ/MYH11+ patients (n=2) clustered together in all clustering analysis performed (using CpG in/out CGIs or all CpG regardless of location). None of the other molecular abnormalities (i.e. DNMT3A mutations, MLL translocations, NPM1 mutations, FTL3/ITDs and CEPBα mutations) formed clusters. We did not observe any effect of clinical status of AML (de novo, secondary AML with dysplastic changes or relapsed AML). Based on this observation, we extracted genomic regions being uniquely differentially methylated for CBFβ/MYH11+ patients. There was a clear tendency towards hypomethylation in 125 regions out of 182 (69%) displaying lower DNA methylation levels compared to CD34+ pool of controls. All CBFβ/MYH11 differentially methylated regions (DMRs) were uploaded to GREAT (http://bejerano.stanford.edu/great/public/html) and enrichment for genes described as upregulated in inv(16) AML (CBFβ/MYH11+) was seen (reported in Valk et al., NEJM 2004, ID: VALK_AML_CLUSTER_9). This enrichment set comprised 10 genomic regions assigned for 6 genes – MN1, SPARC, ST18, DHRS3, FAM171A and BAHCC1.MN1, SPARC, ST18 and DHRS3 – were chosen for hypomethylation verification by 454 bisulfite pyrosequencing. Altogether 11 CBFβ/MYH11+ (inv(16)), 11 non-inv(16) AML M4, 6 other AML subtypes and 5 healthy control samples were examined by 454 pyrosequencing. Average levels of DNA methylation for selected regions were significantly lower for inv(16) versus non-inv(16) AML M4, other AML subtypes and healthy controls (P<0.0001). The 454 results support our theory about site specific CBFβ/MYH11 hypomethylation signature. MN1 gene was overexpressed in 82% of inv(16) AML versus 42% of non-inv(16) patients (χ2 test, P=0.009). Interestingly, MN1 overexpression in non-inv(16) patients was not accompanied with MN1 hypomethylation suggesting different mechanisms of gene deregulation in various AML subtypes.We found a new hypomethylation signature in CBFβ/MYH11+ patients. The described hypomethylated regions were assigned to genes known as being overexpressed in inv(16) patients. As for MN1 gene, we can speculate that different mechanism responsible for its overexpression in inv(16) versus non-inv(16) patients may explain discrepancy between its negative prognostic value in cytogenetically normal AML patients and generally good outcome of inv(16) patients.This study was supported by the project (Ministry of Health, Czech Republic) for conceptual development of research organization (00023736, IHBT) and was a part of the COST Action BM0801 (EuGESMA). Disclosures:No relevant conflicts of interest to declare.

Common Fusion Transcripts Identified in Colorectal Cancer Cell Lines by High-Throughput RNA Sequencing

Colorectal cancer (CRC) is the third most common cancer disease in the Western world, and about 40% of the patients die from this disease. The cancer cells are commonly genetically unstable, but only a few low-frequency recurrent fusion genes have so far been reported for this disease. In this study, we present a thorough search for novel fusion transcripts in CRC using high-throughput RNA sequencing. From altogether 220 million paired-end sequence reads from seven CRC cell lines, we identified 3391 candidate fused transcripts. By stringent requirements, we nominated 11 candidate fusion transcripts for further experimental validation, of which 10 were positive by reverse transcription-polymerase chain reaction and Sanger sequencing. Six were intrachromosomal fusion transcripts, and interestingly, three of these, AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2, were present in, respectively, 18, 18, and 20 of 21 analyzed cell lines and in, respectively, 18, 61, and 48 (17%-58%) of 106 primary cancer tissues. These three fusion transcripts were also detected in 2 to 4 of 14 normal colonic mucosa samples (14%–28%). Whole-genome sequencing identified a specific genomic breakpoint in COMMD10-AP3S1 and further indicates that both the COMMD10-AP3S1 and AKAP13-PDE8A fusion transcripts are due to genomic duplications in specific cell lines. In conclusion, we have identified AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2 as novel intrachromosomal fusion transcripts and the most highly recurring chimeric transcripts described for CRC to date. The functional and clinical relevance of these chimeric RNA molecules remains to be elucidated.

Identification of SNP markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae) using RAD sequencing

Phylogenetic relationships among temperate species of bamboo are difficult to resolve, owing to both the challenge of detecting sufficiently variable markers and their polyploid history. Here, we use restriction site-associated DNA sequencing to identify candidate loci with fixed allelic differences segregating between and within two temperate species of bamboos: Arundinaria faberi and Yushania brevipaniculata. Approximately 27 million paired-end sequencing reads were generated across four samples. From pooled data, we assembled 67685 and 70668 de novo contigs from partial overlap among paired-end reads, with an average length of 240 and 241bp for the two species, respectively, which were used to investigate functional classification of RAD tags in a blastx search. Analysed separately by population, we recovered 29443 putatively orthologous RAD tags shared across the four sampled populations, containing 28023 sequence variants, of which c. 13000 are segregating between species, and c. 3000 segregating between populations within each species. Analyses based on these RAD tags yielded robust phylogenetic inferences, even with data set constructed from surprisingly few loci. This study illustrates the potential for reduced-representation genome data to resolve difficult phylogenetic relationships in temperate bamboos.

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

BackgroundThe 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.ResultsWe 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.ConclusionsDespite 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.

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.

Identification, reconstruction and validation of insertions in resequencing projects with GapFiller

Motivations. A difficult problem in resequencing projects is the identification, reconstruction, and validation of inserted regions within an unknown genome, with respect to a reference one. In most organisms, many structural variants are found in highly repetitive regions of the genome, making their identification difficult. Recent studies demonstrate the feasibility of detecting structural variants using next-generation, paired-end sequencing reads. Reconstructing completely or at least both ends straddling the inserted sequence is a first validation step. Both reconstruction and validation are still weak in existing tools. Methods. GapFiller is a tool developed to fill with a single sequence the gap between paired reads produced by NGS technologies. It is based on a seed-and-extend scheme and it implements techniques to avoid errors in produced contigs. A contig spanning the whole gap is dubbed certified if the mate of the seed read is found. As a matter of fact, GapFiller can be applied to whatever pair of sequences known to lay at an estimated distance, as long as a set of uniformly distributed short reads are provided as input to fill the gap. Our pipeline to reconstruct and validate insertions in a resequenced individual is divided in two main phases: the first one consists in constructing contigs straddling the borders of putative insertions, the second one in filling the gap between them. Before running the pipeline we need to determine locations of putative insertions and to extract the reads aligning around them. More specifically, if insertions in organism A with respect to organism B are to be investigated, we first extract locations of putative insertions, using a tool designed for this purpose (i.e., BreakDancer). Then the reads of A are aligned against the reference B and those mapping next to insertions are extracted, as well as their, possibly unmapped, mates, with the proper orientation. GapFiller is then run twice: a first time to fill the gap between the extracted paired reads in order to reconstruct the borders of each insertion, and a second time to reconstruct the sequence between the certified contigs produced. In the latter phase we treat contigs on the left and on the right side of an insertion as if they were the two pairs of a paired read, respectively, and GapFiller's output will be a super-contig. The event that the super-contig obtained starting from the left contig finally matches against the right one, represents an evidence that we have reconstructed the desired sequence. Clearly, the level of confidence is a function of the number of super-contigs for each (putative) insertion. Moreover, as an important byproduct, we have the assembly of the missing sequence. Results. In order to check correctness we tested our pipeline on a real dataset, consisting of a 30x coverage of paired reads from the Vitis vinifera variety PN40024 (485Mbp), for which the reference genome is known. Using BreakDancer we extracted pairs of coordinates on the reference corresponding to deletions on the resequenced variety Sangiovese. This way we simulated insertions in PN40024, with the advantage of being able to check if the sequences assembled by GapFiller were correct, by simply aligning them against the reference. As a preliminar validation step, for each sequence S identifying a putative insertion we computed the maximal tails of S covered by the (certified) contigs produced in the first phase, i.e. we consider only the contigs consisting of paired reads whose gap has been successfully filled. In particular, we identified 800 putative insertions for which we were able to correctly assemble at least 200bp on both tails. The second phase is more difficult as we try to reconstruct (probably highly) repetitive regions. For a few pairs of left and right contigs we were able to entirely reconstruct the inserted sequence, with respect to the reference genome PN40024. However, this point requires a deeper analysis and we are currently working on the improvement of the final step of our pipeline.

Abstract SY25-01: Analysis of next-generation sequencing data for cancer genomes: challenges and pitfalls

Abstract The characterization of the landscape of genetic lesions that underlie cancer has been significantly advanced with the recent application of next-generation sequencing (NGS) technology. This methodology can be used to sequence selected subsets of genes, the whole exome, the whole genome, or the expressed transcriptome within a cancer cell. By comparing the acquired sequences from both a cancer and matched normal tissue sample from the same patients, one should be able to identify almost all somatic lesions within the cancer. As part of the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project (PCGP), we have undertaken the approach of performing whole genome sequencing (WGS) on 600 pediatric cancers and matched control tissue (1200 total genomes). Although the acquisition of the primary sequence is a formidable challenge, the analysis of these data is where the real work begins. Unfortunately, the majority of published NGS analysis methods were developed to identify germ line variation and therefore perform sub-optimally when applied to the task of identifying somatic mutations in cancer genomes. This is in part a result of the distinct difference in logic that must be used to accurately identify all somatic lesions within a cancer. A cancer genome typically exists within a heterogenous DNA sample that is composed of normal cells admixed with an oligoclonal tumor sample. Moreover, the range of somatic lesions seen in cancer is broader than what is seen as part of germ line genetic variation, with some cancers having exceedingly complex genomes containing focal insertions, deletions, inversions, intra-chromosomal and inter-chromosomal rearrangements and large copy number abnormalities. The accurate identification of these lesions requires not only the presence of the lesions within the cancer DNA, but also their absence from the matched germ line sample. To approach these problems, we, as well as others, have recently developed new analytical approaches to enhance our ability to identify the somatic mutations in cancer. The starting point for these analyses is ≥75 bp paired-end sequencing reads from patient matched tumor and normal DNA samples. Our goal is to identify all somatic single-nucleotide variation (SNV), small insertion/deletion (indel), copy number alteration (CNA) and structural variation (SV) that occur within the cancer DNA sample. Paired tumor-normal NGS data were analyzed together to ensure sensitivity for detecting DNA alterations in tumor and for confirming their absence in the matched normal sample. Somatic lesions initially identified by mapped NGS reads were further analyzed using more accurate algorithms to correct errors cause by suboptimal NGS mapping. The sensitivity of the methods we have developed depends on the read depth, but with WGS at 30X haploid coverage we are able to detect mono-allelic mutation present in as low as ∼25% of the analyzed cellular populations. This sensitivity can be significantly enhanced with greater read densities. Key among the methods we have developed are two new algorithms focused on identifying gross DNA alterations: CREST (Clipping REveals STructure) for SV analysis and CONSERTING (COpy Number SEgmentation by Regression Tree) for CNA analysis. CREST uses sequencing reads with partial alignments to the reference human genome (so-called soft-clipped reads) to directly map the breakpoints of somatic SVs. CONSERTING integrates read depth analysis with SV detection and adjust for sequencing artifacts, coverage bias and germ line CNVs. Together, these methods identify somatic lesions with a high validation rate (92-98% of SNV and Indels, 80% for SVs). In this talk, I will highlight the NGS analytic pipeline we have developed and the recent discoveries that have emerged through its application to pediatric cancer genomes. In addition, I will point out some of the significant challenges that remain to be tacked in order for us to identify the full landscape and functional consequences of the somatic mutations in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY25-01. doi:1538-7445.AM2012-SY25-01

Fast and Accurate Taxonomic Assignments of Metagenomic Sequences Using MetaBin

Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp). To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp) with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.

Transcriptome analysis of an invasive weed Mikania micrantha

As an initial step towards understanding the molecular mechanisms by which plants become invasive, we present here the first transcriptome analysis for an invasive weed Mikania micrantha. The analysis was based on the 75-nucleotide short reads data generated by the Illumina Genome Analyzer II system. A total of 31 131 unique sequences were assembled de novo based on 8.7 million filtered paired-end sequence reads for the transcriptome of an individual M. micrantha growing in the field. 73 % of the unique sequences showed significant similarity to existing proteins in the NCBI database, and 21 448 could be grouped based on gene ontology assignments. Of particular interest are the sequences that share homology with genes involved in genome evolution, plasticity, secondary metabolism and defense responses.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

BackgroundWhole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced.ResultsUsing these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways.ConclusionsSequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

Transcriptome-scale homoeolog-specific transcript assemblies of bread wheat

BackgroundBread wheat is one of the world’s most important food crops and considerable efforts have been made to develop genomic resources for this species. This includes an on-going project by the International Wheat Genome Sequencing Consortium to assemble its large and complex genome, which is hexaploid and contains three closely related ‘homoeologous’ copies for each chromosome. This multi-national effort avoids the complications polyploidy entails for correct assembly of the genome by sequencing flow-sorted chromosome arms one at a time. Here we report on an alternate approach, a direct homoeolog-specific assembly of the expressed portion of the genome, the transcriptome.ResultsAfter assessment of the ability of various assemblers to generate homoeolog-specific assemblies, we employed a two-stage assembly process to produce a high-quality assembly of the transcriptome of hexaploid wheat from Roche-454 and Illumina GAIIx paired-end sequence reads. The assembly process made use of a rapid partitioning of expressed sequences into homoeologous clusters, followed by a parallel high-fidelity assembly of each cluster on a 1150-processor compute cloud. We assessed assembly quality through comparison to known wheat gene sequences and found that in ca. 98.5% of cases the assembly was sufficiently accurate for homoeologous triplets to be cleanly separated into either two or three separate contigs. Comparison to publicly available transcript collections suggests that the assembly covers ~75-80% of the complete transcriptome.ConclusionsThis work therefore describes the first homoeolog-specific sequence assembly of the wheat transcriptome and provides a reference transcriptome for future wheat research. Furthermore, our assembly methodology is transferable to other polyploid organisms.

A Genome Sequence Resource for the Aye-Aye (Daubentonia madagascariensis), a Nocturnal Lemur from Madagascar

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ∼3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ∼2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ∼10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage—a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests.

Transcriptome analysis of an invasive weed Mikania micrantha

As an initial step towards understanding the molecular mechanisms by which plants become invasive, we present here the first transcriptome analysis for an invasive weed Mikania micrantha. The analysis was based on the 75-nucleotide short reads data generated by the Illumina Genome Analyzer II system. A total of 31 131 unique sequences were assembled de novo based on 8.7 million filtered paired-end sequence reads for the transcriptome of an individual M. micrantha growing in the field. 73 % of the unique sequences showed significant similarity to existing proteins in the NCBI database, and 21 448 could be grouped based on gene ontology assignments. Of particular interest are the sequences that share homology with genes involved in genome evolution, plasticity, secondary metabolism and defense responses.

Abstract LB-249: Oncogene-mediated alterations in chromatin conformation

Abstract Emerging evidence suggests that the three-dimensional structure of chromosomes is not random but rather organized into compartments of open and closed chromatin and that this architectural organization plays a major role in gene regulation and genomic aberrations. There is evidence that transcription factors mediate chromatin topology and have been implicated in DNA breaks leading to genomic rearrangement. However, no study, to date, has described a comprehensive, genome-wide characterization of chromatin topological changes, gene regulation, and genomic alterations that can be attributed to oncogenic transcription factors. ETS rearranged prostate cancers, similar to other translocation tumors, represent a common and distinct molecular subclass of PCa based on studies demonstrating characteristic morphologic features, natural history and specific genomic and expression profiles. We hypothesize that ERG mediates specific alterations in chromatin structure that translates into changes in gene expression and genomic alterations in prostate cancer. We used next-generation DNA sequencing and chromosome conformation capture technology (Hi-C) to systematically map long-range chromatin interactions in the prostate cancer genomes of isogenic cell lines that differ in respect to ERG over-expression. Two biological replicate HiC samples were sequenced using five lanes of an Illumina GAIIx flow cell per cell line. After filtering, we obtained a total of roughly 20 million paired end sequence reads that corresponded to intra- and interchromosome interactions per cell line. We validated specific long-range interactions using targeted PCR from 3C libraries and using fluorescent in situ hybridization (FISH). In addition, we performed chromatin-immunoprecipitation with DNA sequencing (ChIPseq) and next generation RNA sequencing (RNA-seq) on the same cell lines to evaluate areas of ERG binding, determine the landscape of histone marks and quantify differential mRNA expression. We identified chromosomal interactions that appear to be dependent on ERG expression and these interactions are associated with ERG binding and changes in gene expression. Finally, we performed an integrative analysis of available whole genome DNA sequencing data (30 x coverage) from 7 prostate cancer samples (Berger et al., Nature 2011) and found associations between mapped genomic rearrangement, ERG binding and cell line specific long range chromosome interactions. Our study indicates, for the first time, that oncogenic transcription factors exert a broad effect on chromatin topology and associated gene regulation, and suggest that chromatin conformation predisposes the genome for later rearrangement events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-249. doi:10.1158/1538-7445.AM2011-LB-249

Global Identification of Genomic Structural Variants In Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias by Mate-Pair Massively Parallel Sequencing.

AbstractAbstract 3364 IntroductionChimeric fusion genes generated by chromosomal translocations are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and mainly represent early, prenatal events. The t(12;21)(p13;q22) translocation generates the ETV6/RUNX1 fusion gene and is the most frequent gene recombination in childhood B-lineage ALL, occurring in approximately 25% of the cases. It is associated with favorable prognosis even though a substantial proportion of the cases relapse. The fusion gene itself initiates the pre-leukemic process but additional genetic hits are needed to trigger a full blown leukemia. Being one of the best-characterized childhood leukemias, both nature and mechanisms of the events that cooperate with the chimeric protein are still poorly understood. Furthermore, studies addressing the genetic origin of relapse demonstrated a clonal relationship between relapse and diagnostic sample, assuming the existence of an ancestral, pre-leukemic clone. ObjectiveSecond-generation sequencing of both ends of huge numbers of DNA fragments allows comprehensive characterization of patterns of somatic rearrangements on an unprecedented, high-resolution level. By using the Illumina mate-pair massively parallel sequencing technology and intra-individual side-by-side comparison of leukemic and normal germline DNA, we aim to elucidate the cooperating genetic events in leukemogenesis in ETV6/RUNX1-ALLs as well as the clonal relationship between relapse and diagnostic sample. MethodsWe investigated diagnostic and relapse samples as well as non-leukemic germline material from one pediatric patient, diagnosed with ETV6/RUNX1-ALL in Germany. Mate-pair genomic sequencing libraries with an insert size of approximately 2-kb were constructed and paired-end sequence reads of 36-bp each were generated on the Illumina Genome Analyzer IIx from randomly created ~500-bp DNA fragments. Data were filtered and aligned to the human reference genome (GRCh37) using BWA. Reads considered PCR duplicates were removed and detection as well as clustering of structural variants (translocations, deletions, inversions) was subsequently carried out with GASV. Discordantly mapping read pairs defined potential structural variations and cluster sizes of at least 4 uniquely and correctly mapping read pairs were included in further analyses. In order to confirm breakpoints and resolve them to base-pair level, areas of putative chromosomal rearrangements were amplified from genomic DNA of tumor and matched normal sample and were conventionally sequenced. ResultsAn average of ~73,000,000 read pairs were generated for each sample and after alignment, the whole genome was sequenced with a mean fragment coverage of 18.8X. A substantial variation in prevalence of structural variants could be detected between paired diagnostic and relapse samples. Within the diagnostic sample we could in total observe 739 deletions, 66 inversions and 107 translocations while the relapse sample exhibited 26 deletions, 14 inversions and 240 translocations. In a first analysis we focused on translocations, presuming the high impact of chromosomal rearrangements on leukemogenesis. Subtracting translocations being of germline origin, 73 translocations at diagnosis and 207 translocations in relapse could be detected, of which 183 (74%) were identified being intragenic. Remarkably, both samples shared only 16 translocations (6%), while 57 (22%) uniquely appear in the diagnostic sample and 191 (72%) could only be observed in the relapse sample. Intragenic, shared translocations in diagnostic and relapse samples include the t(12;24) PDE3A/RN18S1, the t(2;17) PID1/UNC45B as well as the t(1;6) HFM1/EYA4 fusion gene products. Detection of the known ETV6/RUNX1 translocation in diagnostic and relapse sample as well as confirmation of selected breakpoints via Sanger sequencing validated our methodological approach. ConclusionMate-pair sequencing of leukemic samples in comparison to germline material provides a powerful tool to identify genome-wide chromosomal structural variations and will allow analysis of clonality between diagnostic and relapse samples. The low percentage of shared translocations gives a first hint probably objecting the thesis of a common pre-leukemic clone, but will only be elucidated by analysis of further patient samples. Disclosures:No relevant conflicts of interest to declare.

Using paired-end sequences to optimise parameters for alignment of sequence reads against related genomes

BackgroundThe advent of cheap high through-put sequencing methods has facilitated low coverage skims of a large number of organisms. To maximise the utility of the sequences, assembly into contigs and then ordering of those contigs is required. Whilst sequences can be assembled into contigs de novo, using assembled genomes of closely related organisms as a framework can considerably aid the process. However, the preferred search programs and parameters that will optimise the sensitivity and specificity of the alignments between the sequence reads and the framework genome(s) are not necessarily obvious. Here we demonstrate a process that uses paired-end sequence reads to choose an optimal program and alignment parameters.ResultsUnlike two single fragment reads, in paired-end sequence reads, such as BAC-end sequences, the two sequences in the pair have a known positional relationship in the original genome. This provides an additional level of confidence over match scores and e-values in the accuracy of the positional assignment of the reads in the comparative genome. Three commonly used sequence alignment programs: MegaBLAST, Blastz and PatternHunter were used to align a set of ovine BAC-end sequences against the equine genome assembly. A range of different search parameters, with a particular focus on contiguous and discontiguous seeds, were used for each program. The number of reads with a hit and the number of read pairs with hits for the two end sequences in the tail-to-tail paired-end configuration were plotted relative to the theoretical maximum expected curve. Of the programs tested, MegaBLAST with short contiguous seed lengths (word size 8-11) performed best in this particular task. In addition the data also provides estimates of the false positive and false negative rates, which can be used to determine the appropriate values of additional parameters, such as score cut-off, to balance sensitivity and specificity. To determine whether the approach also worked for the alignment of shorter reads, the first 240 bases of each BAC end sequence were also aligned to the equine genome. Again, contiguous MegaBLAST performed the best in optimising the sensitivity and specificity with which sheep BAC end reads map to the equine and bovine genomes.ConclusionsPaired-end reads, such as BAC-end sequences, provide an efficient mechanism to optimise sequence alignment parameters, for example for comparative genome assemblies, by providing an objective standard to evaluate performance.

BreakDancer: an algorithm for high-resolution mapping of genomic structural variation

Detection and characterization of genomic structural variation are important for understanding the landscape of genetic variation in human populations and in complex diseases such as cancer. Recent studies demonstrate the feasibility of detecting structural variation using next-generation, short-insert, paired-end sequencing reads. However, the utility of these reads is not entirely clear, nor are the analysis methods under which accurate detection can be achieved. The algorithm BreakDancer predicts a wide variety of structural variants including indels, inversions, and translocations. We examined BreakDancer's performance in simulation, comparison with other methods, analysis of an acute myeloid leukemia sample, and the 1,000 Genomes trio individuals. We found that it substantially improved the detection of small and intermediate size indels from 10 bp to 1 Mbp that are difficult to detect via a single conventional approach.

Statistical aspects of discerning indel-type structural variation via DNA sequence alignment

BackgroundStructural variations in the form of DNA insertions and deletions are an important aspect of human genetics and especially relevant to medical disorders. Investigations have shown that such events can be detected via tell-tale discrepancies in the aligned lengths of paired-end DNA sequencing reads. Quantitative aspects underlying this method remain poorly understood, despite its importance and conceptual simplicity. We report the statistical theory characterizing the length-discrepancy scheme for Gaussian libraries, including coverage-related effects that preceding models are unable to account for.ResultsDeletion and insertion statistics both depend heavily on physical coverage, but otherwise differ dramatically, refuting a commonly held doctrine of symmetry. Specifically, coverage restrictions render insertions much more difficult to capture. Increased read length has the counterintuitive effect of worsening insertion detection characteristics of short inserts. Variance in library insert length is also a critical factor here and should be minimized to the greatest degree possible. Conversely, no significant improvement would be realized in lowering fosmid variances beyond current levels. Detection power is examined under a straightforward alternative hypothesis and found to be generally acceptable. We also consider the proposition of characterizing variation over the entire spectrum of variant sizes under constant risk of false-positive errors. At 1% risk, many designs will leave a significant gap in the 100 to 200 bp neighborhood, requiring unacceptably high redundancies to compensate. We show that a few modifications largely close this gap and we give a few examples of feasible spectrum-covering designs.ConclusionThe theory resolves several outstanding issues and furnishes a general methodology for designing future projects from the standpoint of a spectrum-wide constant risk.

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

BackgroundWith a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library.ResultsAn initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly.ConclusionThese results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.

Paired-end sequence mapping detects extensive genomic rearrangement and translocation during divergence of Francisella tularensis subsp. tularensis and Francisella tularensis subsp. holarctica populations.

Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR(holar)(c)(tica)) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR(holarctica) are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica, implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica. PCR analysis of the CR(holarctica) in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR(holarctica) are highly conserved, particularly in F. tularensis subsp. holarctica, consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis-like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.