SoftSearch: Integration of Multiple Sequence Features to Identify Breakpoints of Structural Variations

<div><p>Background</p><p>Structural variation (SV) represents a significant, yet poorly understood contribution to an individual’s genetic makeup. Advanced next-generation sequencing technologies are widely used to discover such variations, but there is no single detection tool that is considered a community standard. In an attempt to fulfil this need, we developed an algorithm, SoftSearch, for discovering structural variant breakpoints in Illumina paired-end next-generation sequencing data. SoftSearch combines multiple strategies for detecting SV including split-read, discordant read-pair, and unmated pairs. Co-localized split-reads and discordant read pairs are used to refine the breakpoints. </p> <p>Results</p><p>We developed and validated SoftSearch using real and synthetic datasets. SoftSearch’s key features are 1) not requiring secondary (or exhaustive primary) alignment, 2) portability into established sequencing workflows, and 3) is applicable to any DNA-sequencing experiment (e.g. whole genome, exome, custom capture, etc.). SoftSearch identifies breakpoints from a small number of soft-clipped bases from split reads and a few discordant read-pairs which on their own would not be sufficient to make an SV call. </p> <p>Conclusions</p><p>We show that SoftSearch can identify more true SVs by combining multiple sequence features. SoftSearch was able to call clinically relevant SVs in the BRCA2 gene not reported by other tools while offering significantly improved overall performance.</p> </div

Overlap of true positive calls for the NA12878 and NA18507 datasets.

<p>Overlap of true positive calls for the NA12878 and NA18507 datasets.</p

The general strategy for SoftSearch.

<p>A) Left clipped reads are defined as where the clipped portion of the read is at a smaller genome coordinate than the opposite end (opposite for right clipping). For a left clipped read located on the “+” strand, SoftSearch looks upstream for a discordant read pair where the read is oriented in the “-” direction. The orientation and location of the mate is where SoftSearch links the first region to. To increase the likelihood of exactly detecting the breakpoint, it then looks upstream for a right clipped read cluster. If none is found, then the default breakpoint location is the discordant read mate location; otherwise it is the position of soft clipping at the right clipped read. B) SoftSearch determines discordant read pairs by their insert size and orientation and places them in a temporary BAM file. It also reads the input BAM file for soft clipped reads and converts them to a BED file. Overlapping soft clip locations are counted to identify putative breakpoints, and then queried against the discordant read pair bam file for properly oriented supporting reads, which are then output in VCF format.</p

Example IGV screenshot of a 71bp tandem duplication in the BRCA2 gene identified by SoftSearch.

<p>Discordant reads are blue (plus strand) or red (minus strand). Soft clipped bases appear as multicolour “rainbows”.</p

Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma

<div><p>Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the <i>FGFR2</i> locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (<i>in vitro</i> FGFR2 IC₅₀≈350 nM) was noted in a patient with an <i>FGFR2-TACC3</i> fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (<i>in vitro</i>, FGFR2 IC₅₀≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in <i>ERRFI1</i>, a direct negative regulator of <i>EGFR</i> activation. Rapid and robust disease regression was noted in this <i>ERRFI1</i> inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. <i>FGFR2</i> fusions and <i>ERRFI</i> mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.</p></div

Copy number changes and structural rearrangements.

<p>Whole genome data was utilized to determine copy number alterations and structural rearrangements in the genome for Patients 1–5. WGS was not conducted for patient 6. Red indicates copy number gain, green copy number loss and blue lines indicate structural rearrangements. Significant variability between samples was observed for both copy number changes and structural rearrangements. Patient 5 presented with numerous copy number changes and structural rearrangements contrasting with patient 4 who had minimal structural rearrangements and much smaller regions of copy number changes. Patient 3 is characterized by a large number of structural rearrangements with almost no copy number alterations; in contrast, Patient 1 has a moderate number of structural variations, but has large regions of copy number gain and loss. Patient 2 has a moderate number of structural rearrangements with multiple focal amplifications across the genome.</p

Anti-tumor activity of Patient 3, harboring an <i>ERRFI1</i> mutation, to erlotinib, an EGFR inhibitor.

<p><b>A</b>) CT images of patient 3 at baseline and three months demonstrate significant tumor shrinkage (red marks). CT demonstrates right retroperitoneal lymph nodes decreasing from 7.6 cm to 2.9 cm and left retroperitoneal lymph nodes decreasing from 3.3 cm to 1.7 cm. <b>B</b>) PET images of patient 3 at baseline and three months demonstrate significant tumor shrinkage (red arrows). Hypermetabolic areas corresponding to right retroperitoneal lymph nodes demonstrate decrease from 8 cm longest diameter to imperceptible and left retroperitoneal lymph nodes decreasing from 4.2 cm to 1.4 cm. Both regions demonstrated significant reduction in metabolic activity.</p

Fusion events.

<p>Fusion events.</p

Immunohistochemistry demonstrating pFRS2 Y436, and pERK expression in Patients 1, 4, 5 and 6.

<p><b>A</b>) Tumor stained with pFRS2 Y436 antibody. Patient 1 tumor cells demonstrating both strong cytoplasmic and nuclear expression of pFRS2 (solid arrows); background fibrous stroma is negative (empty arrows). Patient 4 tumor cells show strong nuclear expression and moderate to strong cytoplasmic positivity (solid arrows); occasional background fibrous stromal cells are negative for pFRS2 (empty arrows) and scattered tumor cells show basolateral/membranous staining as well (white arrows). Patient 5 tumor cells show intensely strong expression in both nuclei and cytoplasm (solid arrows); scattered background fibrous stromal cells are negative (empty arrows). Patient 6 tumor cells show negative nuclear expression of pFRS2, moderate cytoplasmic expression and basolateral or membranous expression of varying intensity (solid arrows); background fibrous stromal cells are negative (empty arrows). <b>B</b>) Tumor stained with pERK(MAPK) antibody. Patient 1 demonstrates negative/weak fibrous stroma (empty arrows) and tumor cells with negative nuclei and moderate to strong cytoplasmic expression (solid arrows). Patient 4 demonstrates negative inflammatory background (empty arrows) tumor cells with variable negative to strong nuclear expression and moderate to strong cytoplasmic positivity (solid arrows). Patient 5 demonstrates negative/weak fibrous stroma (empty arrows) and tumor cells with strong nuclear and cytoplasmic expression (solid arrows). Patient 6 demonstrates negative background lymphocytes/mononuclear inflammatory cells (empty arrows) and tumor cells with strong nuclear and cytoplasmic expression (solid arrows).</p

Sequence variation effects.

<p>Functional effects of high confidence sequence variations for all of the patients were identified as described in the Methods. The abundance of variations in each functional category is provided as percentages relative to the total number of high confidence variations and raw counts are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004135#pgen-1004135-t001" target="_blank"><b>Table 1</b></a>. For categories where the percentage was less than 5%, values are not shown. Summaries by individual patients are shown as follows: <b>A</b>) Patient 1, <b>B</b>) Patient 2, <b>C</b>) Patient 3, <b>D</b>) Patient 4, <b>E</b>) Patient 5, and <b>F</b>) Patient 6. Nonsynonymous single nucleotide variations were the predominant class in all of the patients. Two patients, Patients 1 and 2 also accumulated a high number of synonymous mutations in comparison to the other patients; Patient 5 carries the most stops gained likely contributing to a higher number of pseudogenes in comparison to the others; Patient 5 was also the only patient to carry several predicted high impact mutations that affect the splice site acceptor regions (light green, percentage <5%). In addition to the major functional classes summarized, Patient 6 also carried a codon change plus insertion variation.</p