Analysis Of Whole Genome Sequencing Data Research Articles

Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes.

Retrotransposition of gene transcripts leads to structural variation in mammalian genomes

BackgroundRetroposed processed gene transcripts are an important source of material for new gene formation on evolutionary timescales. Most prior work on gene retrocopy discovery compared copies in reference genome assemblies to their source genes. Here, we explore gene retrocopy insertion polymorphisms (GRIPs) that are present in the germlines of individual humans, mice, and chimpanzees, and we identify novel gene retrocopy insertions in cancerous somatic tissues that are absent from patient-matched non-cancer genomes.ResultsThrough analysis of whole-genome sequence data, we found evidence for 48 GRIPs in the genomes of one or more humans sequenced as part of the 1,000 Genomes Project and The Cancer Genome Atlas, but which were not in the human reference assembly. Similarly, we found evidence for 755 GRIPs at distinct locations in one or more of 17 inbred mouse strains but which were not in the mouse reference assembly, and 19 GRIPs across a cohort of 10 chimpanzee genomes, which were not in the chimpanzee reference genome assembly. Many of these insertions are new members of existing gene families whose source genes are highly and widely expressed, and the majority have detectable hallmarks of processed gene retrocopy formation. We estimate the rate of novel gene retrocopy insertions in humans and chimps at roughly one new gene retrocopy insertion for every 6,000 individuals.ConclusionsWe find that gene retrocopy polymorphisms are a widespread phenomenon, present a multi-species analysis of these events, and provide a method for their ascertainment.

Rainbow: a tool for large-scale whole-genome sequencing data analysis using cloud computing.

BackgroundTechnical improvements have decreased sequencing costs and, as a result, the size and number of genomic datasets have increased rapidly. Because of the lower cost, large amounts of sequence data are now being produced by small to midsize research groups. Crossbow is a software tool that can detect single nucleotide polymorphisms (SNPs) in whole-genome sequencing (WGS) data from a single subject; however, Crossbow has a number of limitations when applied to multiple subjects from large-scale WGS projects. The data storage and CPU resources that are required for large-scale whole genome sequencing data analyses are too large for many core facilities and individual laboratories to provide. To help meet these challenges, we have developed Rainbow, a cloud-based software package that can assist in the automation of large-scale WGS data analyses.ResultsHere, we evaluated the performance of Rainbow by analyzing 44 different whole-genome-sequenced subjects. Rainbow has the capacity to process genomic data from more than 500 subjects in two weeks using cloud computing provided by the Amazon Web Service. The time includes the import and export of the data using Amazon Import/Export service. The average cost of processing a single sample in the cloud was less than 120 US dollars. Compared with Crossbow, the main improvements incorporated into Rainbow include the ability: (1) to handle BAM as well as FASTQ input files; (2) to split large sequence files for better load balance downstream; (3) to log the running metrics in data processing and monitoring multiple Amazon Elastic Compute Cloud (EC2) instances; and (4) to merge SOAPsnp outputs for multiple individuals into a single file to facilitate downstream genome-wide association studies.ConclusionsRainbow is a scalable, cost-effective, and open-source tool for large-scale WGS data analysis. For human WGS data sequenced by either the Illumina HiSeq 2000 or HiSeq 2500 platforms, Rainbow can be used straight out of the box. Rainbow is available for third-party implementation and use, and can be downloaded from http://s3.amazonaws.com/jnj_rainbow/index.html.

Two Different High Throughput Sequencing Approaches Identify Thousands of De Novo Genomic Markers for the Genetically Depleted Bornean Elephant

High throughput sequencing technologies are being applied to an increasing number of model species with a high-quality reference genome. The application and analyses of whole-genome sequence data in non-model species with no prior genomic information are currently under way. Recent sequencing technologies provide new opportunities for gathering genomic data in natural populations, laying the empirical foundation for future research in the field of conservation and population genomics. Here we present the case study of the Bornean elephant, which is the most endangered subspecies of Asian elephant and exhibits very low genetic diversity. We used two different sequencing platforms, the Roche 454 FLX (shotgun) and Illumina, GAIIx (Restriction site associated DNA, RAD) to evaluate the feasibility of the two methodologies for the discovery of de novo markers (single nucleotide polymorphism, SNPs and microsatellites) using low coverage data. Approximately, 6,683 (shotgun) and 14,724 (RAD) SNPs were detected within our elephant sequence dataset. Genotyping of a representative sample of 194 SNPs resulted in a SNP validation rate of ∼ 83 to 94% and 17% of the loci were polymorphic with a low diversity (H o = 0.057). Different numbers of microsatellites were identified through shotgun (27,226) and RAD (868) techniques. Out of all di-, tri-, and tetra-microsatellite loci, 1,706 loci had sufficient flanking regions (shotgun) while only 7 were found with RAD. All microsatellites were monomorphic in the Bornean but polymorphic in another elephant subspecies. Despite using different sample sizes, and the well known differences in the two platforms used regarding sequence length and throughput, the two approaches showed high validation rate. The approaches used here for marker development in a threatened species demonstrate the utility of high throughput sequencing technologies as a starting point for the development of genomic tools in a non-model species and in particular for a species with low genetic diversity.

Melanoma genome sequencing reveals frequent PREX2 mutations

Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1. To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.

A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff

We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w1118; iso-2; iso-3 strain and the reference y1; cn1 bw1 sp1 strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5′UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5′ and 3′ UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory.

Detection of Novel Mutations In MDS/AML by Whole Genome Sequencing

AbstractAbstract 299Given the poor prognosis of secondary AML evolving from prior MDS and the limited knowledge of mutations that occur during transformation, we set out to comprehensively discover gene mutations that occur during MDS to AML transformation. To achieve this goal, we performed whole genome sequencing (WGS) of paired DNA samples from normal (skin) and tumor (bone marrow) specimens from a patient with MDS-derived AML. The patient was a 65 year old man who presented with pancytopenia and MDS (FAB RA, 4% myeloblasts, del(5q), -17, del(20q)). Three years later he became RBC transfusion dependent and had skin and bone marrow samples banked at Washington University after providing informed consent. His bone marrow biopsy was consistent with RAEB MDS (6% myeloblasts, del(5q), -17, del(20q)), and he subsequently received 4 cycles of decitibine and 2 cycles of lenalidomide before developing AML 2 years later (69% myeloblasts, del(5q), -17, del(20q)). He underwent an allogeneic BMT and died 1 year later. DNA libraries were prepared from the normal (skin) and flow sorted (CD45 dim, low side scatter) AML samples. Using 75–100 bp paired-end reads on the Illumina platform, we generated 126.2 Gb and 101.7 Gb of sequence from the normal and tumor libraries, respectively. Aligned, deduplicated sequence provided 28.7× (normal) and 26.5× (tumor) haploid coverage of the genome, and >96% diploid coverage, using informative SNPs as a metric. Analysis of paired tumor and normal genome sequences allowed us to discriminate between inherited and acquired sequence variants. Indels and structural variants, including copy number alterations, inversions, and translocations were identified using a combination of SAMTools, Pindel, GATK, and Breakdancer algorithms. The cytogenetically visible monosomy 17 and del(20q) lesions were confirmed, and the del(5q) was resolved into 11 distinct interstitial deletions using WGS data. Single nucleotide resolution assembly was possible for 6 of the deleted segments on chromosome 5 and the chromosome 20 deletion which revealed microhomology at the breakpoints, implicating the error prone nonhomologous end joining repair pathway. Interestingly, the tumor suppressor genes APC and DCC were in separate micro-retained regions within larger deletions on chromosome 5, suggesting a selective pressure to retain two copies of these genes during AML progression. Of the 4.8M single nucleotide variants (SNVs) detected in the tumor genome, 33,006 were not found in databases or this patient's normal genome and are, therefore, potential somatic mutations. We identified 46 tier 1 high confidence (HC) SNVs (coding and splice site variants), 388 tier 2 HC SNVs (conserved non-coding variants), 2,185 tier 3 HC SNVs (variants in non-repetitive regions), and 5,862 tier 4 HC SNVs (all other variants). Tier 1 SNVs were prioritized for validation given their potential to be functionally significant. 22/46 Tier 1 HC SNVs were validated as acquired mutations in the AML sample using the 454 platform (5 synonymous, 1 nonsense, and 16 missense mutations). Only 1/22 SNVs has previously been found in myeloid cancers (WT1). The mutant allele frequency of the 22 mutations ranged from 9–100% in the tumor sample based on the 454 deep read counts, suggesting that the underlying heterogeneity that is common in de novo MDS persists during transformation to AML. By sequencing the 22 somatic SNVs in the MDS sample, we identified 10 mutations that were present in both the MDS and AML samples and 12 AML-specific mutations. The latter are likely to be important for MDS to AML evolution. By analyzing the mutant allele frequencies of all 22 SNVs, we predict that 1 dominant clone and 2 sub-clones coexist in the AML sample. All 22 genes with mutations are expressed in the AML sample but none of the 22 SNVs have recurrent mutations at the same nucleotide position in an additional 150 de novo MDS samples. Finally, the residual non-deleted allele of ACTR5, located in the 2.5Mb minimally deleted region (MDR) on chromosome 20, is mutated during AML transformation, implicating it as a potential 20q tumor suppressor gene. Collectively, analysis of WGS data identified mutations in 21 genes not previously implicated in MDS or AML, identified 12 genes as potential drivers of evolution from MDS to AML, allowed for fine mapping of deletion breakpoints on chromosomes 5 and 20, and identified a potential tumor suppressor on the del(20q) MDR. Disclosures:No relevant conflicts of interest to declare.

Points: Recognising placental steroid sulphatase deficiency

<h3>Abstract</h3> The transformation of chronic lymphocytic leukemia (CLL) to high-grade diffuse large B-cell lymphoma (DLBCL), also called Richter’s Syndrome (RS), is a rare cancer with dismal prognosis. Drug discovery for RS is hampered by the lack of suitable experimental models, and effective therapies remain elusive rendering RS an area of high unmet clinical need. We performed whole genome sequencing (WGS) to interrogate paired CLL and RS samples from 17 patients enrolled in a prospective multicenter Phase 2 clinical trial (CHOP-OR) and we found that subclones affected by mutations in MAPK and PI3K pathways show a high expansion probability during transformation. We also demonstrate for the first time that non-coding mutation clusters in a <i>PAX5</i> enhancer, situated 330kb upstream from the transcription initiation site, correlate with transformation. Finally, we confirm our findings by employing targeted DNA sequencing (TGS) and RNA expression profiling on an extended cohort of 38 patients. <h3>Statement of significance</h3> Through integrated analysis of WGS, TGS and RNA expression data, we identified drivers of transformation not previously implicated in RS, which can be targeted therapeutically and tested in the clinic. Our results have informed the design of a new clinical platform study, which is now open to recruitment in the UK.