Burrows-Wheeler Aligner Research Articles

A novel familial mutation associated with Treacher Collins syndrome: A case report.

Treacher Collins syndrome (TCS) is a type of mandibulofacial dysostosis with incomplete penetrance and high intra- and interfamilial clinical heterogeneity, and it is associated with mutations of treacle ribosome biogenesis factor 1 (TCOF1), and RNA polymerase I and III subunit (POLR1)C and POLR1D genes. In the present case report, a patient with TCS with auricle dysplasia and hearing loss accompanied with intellectual disability is described. Sequence analysis was performed on blood samples from the patient and his father via oligonucleotide-based target capture, followed by next-generation sequencing. Alignment and variant calls were generated using the Burrows-Wheeler Aligner and Genome Analysis Toolkit, followed by bioinformatics analysis of the detected variants. A novel heterozygous mutation, c.911C>T (p.Ser304Leu), was detected in the TCOF1 gene, which was inherited from the father. The father of the patient only suffered from hearing loss. The present report is the first to identify an association between phenotypic variability and TCOF1 gene mutations and thus contributes to our understanding of the association between the genotype and phenotype in patients with TCS and offers clinically relevant information for diagnosis of the syndrome.

Abstract P4-09-02: Somatic tumor profiles of breast cancer in Latinas

Abstract Background: Latinos represent the largest and fastest-growing minority population in the US; breast cancer is the most common cancer and causes the most cancer deaths in this population. Precision medicine is being applied increasingly to guide breast cancer treatment decisions. However, the ability to apply precision medicine for prognosis and targeted treatments for breast cancer in Latinas is limited by insufficient data on somatic mutation and gene expression profiles in available tumor mutation datasets. In The Cancer Genome Atlas (TCGA), one of the largest publicly available tumor mutation datasets, only 31 of ~1100 total breast cancers are Latina. Therefore, we performed whole exome sequencing on matched tumor/normal pairs in 146 tumors from 142 women reporting here on their somatic mutational profile compared to non-Latino women of European ancestry from TCGA. Methods: We enrolled 142 Latinas with invasive breast cancer and obtained blood and tumor tissue (formalin-fixed paraffin embedded). After whole exome sequencing of 146 pairs of tumor (somatic) and normal (germline) samples, high quality data were available for 142 tumor-normal pairs for analysis. Target coverage for normal was ~30X and for tumor was ~100X. Sequence data were aligned using Burrows-Wheeler Aligner, genotype calls were made using GATK and Mutect2 was used to call somatic mutations including base substitutions and small insertions and deletions. We estimated genetic ancestry from the germline genotype data using ADMIXTURE. Results: Age at diagnosis ranged from 31 to 75 years with a median age of 48 years. Histologically, 83% were estrogen-receptor positive, 71 % were progesterone-receptor positive, and 17% were human epidermal growth factor receptor positive; 87% were Stage 1 or 2 at diagnosis. The mean genetic ancestry in our sample was 52.3% European, 41.6% Indigenous American, and 6.2% African. We compared the 30 genes most commonly mutated in breast cancers (e.g., PIK3CA, TP53, MAP3K1, MLL3) in non-Latina Whites in TCGA to our data both overall and by subtype and searched for additional recurrently mutated genes not commonly observed in other populations. Similar to non-Latinos, PIK3CA and TP53 were the most commonly mutated genes; mutations in PIK3CA, GATA3, MAP3K1, and RYR2 were significantly more frequent in Latinas (p-value range from 0.001-0.002). Summary: In general, breast tumors from Latinas have similar rates of somatic mutations in most of the top commonly observed genes in European-ancestry women. Citation Format: Susan Neuhausen, Daniel Schmolze, Linda Steele, Shu Tao, Aaron Adamson, Charleen Adams, Donglei Hu, Scott Huntsman, Jung Byun, Kevin Gardner, Eliseo Perez-Stable, Anna Napoles, Jeffrey Weitzel, Elad Ziv. Somatic tumor profiles of breast cancer in Latinas [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-02.

Genomic Landscape of Chinese Patients with Chronic Lymphocytic Leukemia By Whole-Exome Sequencing

Introduction: Through previous studies using Sanger sequencing and targeted gene sequencing, we found that the frequencies of certain genes in Chinese patients with chronic lymphocytic leukemia (CLL) were different from those in CLL patients from the western countries. However, until now, there are no published whole-exome sequencing (WES) studies in Chinese CLL patients. To better define the genomic landscape of CLL cases in China, we conducted WES studies of CLL cases in our center. Methods: This study include 43 CLL patients, of whom 40 were untreated at the time of sampling. The median age of the 43 patients were 61 years, the male-to-female ratio was 2:1. We isolated peripheral blood mononuclear cells, then CD19 positive cells were purified by using magnetic microbeads in some cases. The buccal cells or the leukocytes that were depleted of CD19 positive cells were used as germline controls. We purified genomic DNA and used the purified DNA for library construction, after which we sequenced the library using the Illumina HiSeq2500 platform. After sequencing, software including Burrows-Wheeler Aligner (BWA) and GATK were used for the bioinformatic analysis. Results: In the 43 Chinese patients with CLL, the median number of somatic mutations was 67 (range:10-128) and the median number of nonsynonymous mutations was 26 (range: 5-55). We found that the number of mutations in patients older than 60 was significantly higher than that in the younger patients. Somatic mutations were detected across 991 different genes, of which 24 genes were identified as drivers in the study from the Dana-Faber Cancer Institute (DFCI). The 24 genes included NOTCH1, SF3B1, MYD88, POT1, TP53 and so on. Seventy-nine genes were recurrently mutated and 13 genes were found to be mutated in 3 or more cases. The most frequently mutated gene was NOCTH1 (6 cases, 14.0%). FBXW7, another gene that is involved in the regulation of the NOTCH1 pathway, was also mutated in 3 cases. IGLL5, MUC4 and MYD88 were also among the most frequently mutated genes, with each of them being mutated in 5 cases (11.6%). We found that the frequencies of mutations in IGLL5 (11.6% vs. 2.2%, P=0.0056), MYD88 (11.6% vs. 3.0%, P=0.0147), BCOR (9.3% vs. 2.2%, P=0.0246), and SF3B1 (4.7% vs. 21.0%, P=0.0084) were significantly different between Chinese CLL patients and patients from western countries. BCOR-mutated patients had more frequent aberrations involving NOTCH1 pathway (NOTCH1 and/or FBXW7 mutation) than BCOR-unmutated patients (3/4 [75.0%] vs. 6/39 [15.4%], P=0.0242) (Figure 1A). Analysis of 10967 tumor samples from the Cancer Genome Atlas (TCGA) program revealed that tumors with BCOR mutations had a higher frequency of NOTCH1 aberrations (NOTCH1 and/or FBXW7 mutation) than those without BCOR mutations (116/349 [33.2%] vs. 722/10618 [6.8%]; odds ratio: 6.824, 95%CI: 5.393-8.634, P<0.0001) (Figure 1B). These data suggest BCOR mutations and NOTCH1 aberration may cooperate in the pathogenesis of CLL and other types of tumors. NFKBIA mutations were identified in 2 cases (Figure 1C) and we found that the mRNA level of NFKBIA was significantly lower in the NFKBIA mutated CLL cells than in the normal B cells (Figure 1D-E); in the MEC-1 cell line, significant activation of NF-кB pathway was observed after knockdown of NFKBIA using siRNA (Figure 1F). Conclusion: Patients with CLL in China and patients with CLL in the western countries have different mutational landscapes. BCOR gene mutations may cooperate with aberrations involving the NOTCH1 pathway in the pathogenesis of CLL. NFKBIA could be a potential tumor suppressor in the pathogenesis of CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

1627. Outbreaks of Klebsiella pneumoniae in Special Care Nurseries (SCN) in Jamaica: Role of Whole-Genome Sequencing

Background Klebsiella pneumoniae is a frequent cause of neonatal sepsis and carries a high mortality rate in lower and middle-income countries (LMICs). From March-November 2015, two Jamaican hospitals experienced K. pneumoniae outbreaks in their Special Care Nurseries (SCNs). New admissions to both SCNs were temporarily halted while additional infection control strategies were implemented. 31 babies were infected, of which 15 died. International collaboration was requested to help investigate if the sepsis cases were nosocomial transmission, repeated introductions from the community, or both using whole-genome sequencingMethodsWe sequenced DNA from 19 outbreak isolates (n = 13 from Hospital A, n = 6 from Hospital B) on an Illumina HiSeq2500 instrument and assembled short-reads using SPAdes. We used ResFinder v3.1.0 to screen resistance genes and assigned MLSTs using in-house scripts. To compare the outbreak isolates, we selected a reference genome from among the assembled isolates, aligned raw reads using the Burrows–Wheeler Aligner (BWA), identified SNPs using GATK UnifiedGenotyper, and removed the recombined regions using Gubbins v2.3.4. We further contextualized the 19 outbreak isolates against a global collection of more than 300 K. pneumoniae genomes.ResultsAll 13 isolates from Hospital A appeared to be from a single source. All were ST45 and encoded blaCTX-M-15, which confers extended-spectrum β-lactam (ESBL) resistance. Five of 6 isolates from Hospital B appeared to be from a separate, single source. These 5 isolates were ST268 and susceptible to most antibiotics. 1 isolate from Hospital B was ST628, encoded blaCTX-M-15, and grouped separately from other Hospital B outbreak isolates. Hospital A and B outbreak isolates formed independent, unique clades within a global K. pneumoniae collection.ConclusionOur findings indicate nosocomial transmission was responsible for both neonatal K. pneumoniae outbreaks, rather than repeat introductions from the community. The main sequence types we detected (ST45 and ST268) are not known pandemic clones and may circulate regionally. Multifaceted infection control measures were implemented for effectively halting outbreaks.Disclosures All authors: No reported disclosures.

A20 Sample preparation for whole-genome next-generation sequencing (NGS) of hepatitis C virus (HCV) routine RNA samples

Next-generation sequencing (NGS) is a technique that can capture the variability of viral populations in transmission studies. The conventional sample preparation for NGS, based on amplicons, is a potential source of errors, derived from the variable affinity of specific primers for different viral variants and from irregular DNA polymerase efficiency. In this context, we propose a more reliable method for viral whole genome sample preparation, starting from nucleic acids obtained and stored with conventional procedures. Our goal was to obtain complete hepatitis C virus (HCV) genome sequences to subsequently perform extensive phylogenetic analyses. Additionally, we aimed to test the effectiveness of nuclease treatment used to remove contaminating host DNA. Nucleic acids were obtained from almost cell-free blood plasma of HCV-infected patients. As a source for Illumina library preparation, double-stranded cDNA was generated using random primers. The HCV genome was not amplified before library preparation, avoiding possible biases derived from unequal copying. To get rid of possible host contaminants in the samples, a DNase treatment step was added. Libraries were paired-end sequenced on the Illumina platform using MiSeq reagent kit v3. After conservative filtering of contaminant human reads by alignment with the human reference genome using Burrows-Wheeler Aligner (BWA), the remaining reads were mapped to the HCV reference genome using BWA. Primary maximum likelihood phylogenetic analyses were performed using ClustalW and IQTREE to infer the phylogenetic relationships of the sequenced samples in the context of complete genome sequences of the same genotype. NGS sample preparation method of HCV from blood plasma was established. Complete genome sequences of HCV could be obtained with variable coverage depending on the viral load of plasma samples. No significant reduction of host DNA proportion in DNase treated samples in comparison to the controls was observed. The new sequences clustered within the Los Alamos National Laboratory database-deposited HCV subtype 4d samples. The method can be used to obtain full-length sequences of HCV from nucleic acid samples not previously planned for NGS. No improvement was observed when DNase pre-treatment of nucleic acids extracted from blood plasma was performed.

Combined immunodeficiency in a patient with c-Rel deficiency

This study reports a homozygous mutation in REL abrogating c-Rel protein expression in a patient with combined immunodeficiency characterized by susceptibility to Mycobacterium tuberculosis, Salmonella, Cryptosporidium, and cytomegalovirus.

Holistic optimization of an RNA-seq workflow for multi-threaded environments.

For many next generation-sequencing pipelines, the most computationally intensive step is the alignment of reads to a reference sequence. As a result, alignment software such as the Burrows-Wheeler Aligner is optimized for speed and is often executed in parallel on the cloud. However, there are other less demanding steps that can also be optimized to significantly increase the speed especially when using many threads. We demonstrate this using a unique molecular identifier RNA-sequencing pipeline consisting of 3 steps: split, align, and merge. Optimization of all three steps yields a 40% increase in speed when executed using a single thread. However, when executed using 16 threads, we observe a 4-fold improvement over the original parallel implementation and more than an 8-fold improvement over the original single-threaded implementation. In contrast, optimizing only the alignment step results in just a 13% improvement over the original parallel workflow using 16 threads. Code (M.I.T. license), supporting scripts and Dockerfiles are available at https://github.com/BioDepot/LINCS_RNAseq_cpp and Docker images at https://hub.docker.com/r/biodepot/rnaseq-umi-cpp/. Supplementary data are available at Bioinformatics online.

Calling known variants and identifying new variants while rapidly aligning sequence data

Whole-genome sequencing studies can identify causative mutations for subsequent use in genomic evaluations. Speed and accuracy of sequence alignment can be improved by accounting for known variant locations during alignment instead of calling the variants after alignment as in previous programs. The new programs Findmap and Findvar were compared with alignment using Burrows-Wheeler alignment (BWA) or SNAP and variant identification using Genome Analysis ToolKit (GATK) or SAMtools. Findmap stores the reference map and any known variant locations while aligning reads and counting reference and alternate alleles for each DNA source. Findmap also outputs potential new single nucleotide variant, insertion, and deletion alleles. Findvar separates likely true variants from read errors and outputs genotype probabilities. Strategies were tested using cattle, human, and a completely random reference map and simulated or actual data. Most tests simulated 10 bulls, each with 10× simulated sequence reads containing 39 million variants from the 1000 Bull Genomes Project. With 10 processors, clock times for processing 100× data were 105 h for BWA, 25 h for GATK, and 11 h for SAMtools but only about 4 h for SNAP, 3 h for Findmap, and 1 h for Findvar. Alignment programs required about the same total memory; BWA used 46 GB (4.6 GB/processor), whereas >10 processors can share the same memory in SNAP and Findmap, which used 40 and 46 GB, respectively. Findmap correctly mapped 92.9% of reads (compared with 92.6% from SNAP and 90.5% from BWA) and had high accuracy of calling alleles for known variants. For new variants, Findvar found 99.8% of single nucleotide variants, 79% of insertions, and 67% of deletions; GATK found 99.4, 95, and 90%, respectively; and SAMtools found 99.8, 12, and 16%, respectively. False positives (as percentages of true variants) were 11% of single nucleotide variants, 0.4% of insertions, and 0.3% of deletions from Findvar; 12, 8.4, and 2.9%, respectively, from GATK; and 37, 1.3, and 0.4%, respectively, from SAMtools. Advantages of Findmap and Findvar are fast processing, precise alignment, more useful data summaries, more compact output, and fewer steps. Calling known variants during alignment allows more efficient and accurate sequence-based genotyping.

Identification of causative variants in patients with non-syndromic hearing loss in the Minnan region, China by targeted next-generation sequencing

Background: Due to extreme genetic heterogeneity, targeted next-generation sequencing (NGS) can be an efficient tool for rapid genetic diagnosis of hereditary non-syndromic hearing loss (NSHL).Aims/Objectives: This study was aiming to identify causative variants in NSHL patients from the Minnan region through targeted NGS.Material and methods: Genomic DNA samples from 90 NSHL subjects were extracted and subjected to SureSelect target enrichment system to capture the entire coding exons and flanking intronic regions of gene GJB2, SLC26A4, and MT-RNR1. The captured DNA was then sequenced by Illumina HiSeq2500. The sequence data was processed and quality-checked using Burrows-Wheeler Alignment, Picard, and GATK, and annotated by SnpEff, SIFT, and GERP++.Results: Twenty-six candidate variants in GJB2, SLC26A4, and MT-RNR1 were detected in 57 of 90 NSHL patients. GJB2 c.109G > A was the most frequent variant, followed by GJB2 c.608T > C and c.235delC. Genetic diagnosis was available for 22 NSHL patients, including 19 patients associated with autosomal recessive NSHL, one patients with autosomal dominant NSHL, and two individuals with mitochondrial disorders.Conclusions and significance: Our targeted NGS analysis added supports for the application of NGS in routine diagnosis and provided an overview of genetic variants with allele frequencies in the deaf population from the Minnan region.

Children with Sickle Cell Anemia and APOL1 Gene Variants Develop Albuminuria Early in Life

Introduction: Gene variants in the apolipoprotein L-1 (APOL1) gene are strong modifiers for the development of chronic kidney disease in individuals of African descent and are associated with progression of renal disease and albuminuria in cross-sectional studies of individuals with sickle cell anemia (SCA). While the association of APOL1 with albuminuria in older SCA patients is established, it is unclear whether participants with APOL1 G1 (rs73885319/ rs6090145) and G2 (rs71785313) variants (Ashley-Koch Br J Hematol 2011; Kormann Br J Haematol 2017) are more likely to develop albuminuria early in life. We hypothesized that individuals with SCA with the APOL1 G1 and G2 variants experience albuminuria at a higher rate and at a younger age than individuals without these APOL1 mutations.Methods: APOL1 G1 (rs73885319/ rs6090145) and G2 (rs71785313) variants were identified from whole genome sequence (WGS) data for individuals with SCA (HbSS or HbSβ0 thalassemia) enrolled in the longitudinal Sickle Cell Clinical Research and Intervention Program (SCCRIP; NCT02098863) (Hankins et al, Pediatr Blood Cancer 2018). WGS data were generated by aligning paired-end 150 bp reads to the GRCh38 human reference using the Burrows-Wheeler Aligner (BWA-ALN v0.7.12) and the GATK best-practices workflow implemented in GATK v3.4.0. We modeled the time to first albuminuria diagnosis defined by abnormal urine albumin:creatinine ratio (>30mg/g). Covariates included age, sex, and hydroxyurea therapy. We considered two risk models. Model 1 defined high risk APOL1 mutation as either APOL1 G1 homozygotes or G2 homozygotes or G1/G2 double heterozygotes. Model 2 was defined additively wherein G1 or G2 homozygotes were assumed to confer more risk compared to G1/G2 double heterozygotes.Results: In 285 individuals with SCA, 93% (n=266) with HbSS and 7% (n=19) with HbSβ0 thalassemia, 14% (n=41) experienced albuminuria at a mean age of 11.4 (±3.3) years. In total, 11% (n=32) of this SCA cohort had an APOL1 mutation; 6% (n=17) were G1/G2 double heterozygote, 4% (n=11) were G1 homozygotes, and 1% (n=4) were G2 homozygotes. Among the 32 participants with an APOL1 mutation, 40% (n=13) had albuminuria as compared to 11% (n=28) of the 253 participants without an APOL1 mutation (OR: 9.85, 95% CI 3.84-25.25). There was also a significant association with albuminuria based on the additive genetic model (OR: 4.38, 95% CI 2.39 -8.02) (Table). In a survival analysis, participants with an APOL1 mutation had a hazard ratio (HR) of 3.75 (95% CI: 1.93-7.29, p<0.0001) associated with time to diagnosis of albuminuria compared with individuals with other non-risk APOL1 genotypes. The mean age for individuals having albuminuria with APOL1 G1/G2 risk alleles was 9.8 (±3.2) years, roughly 2 years younger than those without the risk alleles (11.8 ±4.7 years) (Satterthwaite t value=3.10, p=0.003). APOL1 G1 alleles also contributed individual risk of albuminuria (HR=1.66, p=0.0374).Summary: Our analyses found that pediatric individuals with SCA who have APOL1 two risk alleles are at high risk of developing albuminuria which many will experience early in childhood. This important finding has significant implications for clinical care. First, using a genetic screening program for all patients with SCAC can identify individuals with APOL1 mutations. Second, individuals with SCA and an APOL1 mutation should be screened for albuminuria during childhood. Finally, early initiation of therapeutic strategies to ameliorate renal dysfunction needs to be evaluated in the presence of APOL1 mutations. [Display omitted] DisclosuresHankins:Global Blood Therapeutics: Research Funding; Novartis: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:ASH Scholar: Research Funding; Daiichi Sankyo: Consultancy; NHLBI: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding.

The genomics of desmoplastic small round cell tumor reveals the deregulation of genes related to DNA damage response, epithelial\u2013mesenchymal transition, and immune response

BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare, aggressive, and poorly investigated simple sarcoma with a low frequency of genetic deregulation other than an Ewing sarcoma RNA binding protein 1 (EWSR1)-Wilm’s tumor suppressor (WT1) translocation. We used whole-exome sequencing to interrogate six consecutive pre-treated DSRCTs whose gene expression was previously investigated.MethodsDNA libraries were prepared from formalin-fixed, paraffin-embedded archival tissue specimens following the Agilent SureSelectXT2 target enrichment protocol and sequenced on Illumina NextSeq 500. Raw sequence data were aligned to the reference genome with Burrows–Wheeler Aligner algorithm. Somatic mutations and copy number alterations (CNAs) were identified using MuTect2 and EXCAVATOR2, respectively. Biological functions associated with altered genes were investigated through Ingenuity Pathway Analysis (IPA) software.ResultsA total of 137 unique somatic mutations were identified: 133 mutated genes were case-specific, and 2 were mutated in two cases but in different positions. Among the 135 mutated genes, 27% were related to two biological categories: DNA damage-response (DDR) network that was also identified through IPA and mesenchymal–epithelial reverse transition (MErT)/epithelial–mesenchymal transition (EMT) already demonstrated to be relevant in DSRCT. The mutated genes in the DDR network were involved in various steps of transcription and particularly affected pre-mRNA. Half of these genes encoded RNA-binding proteins or DNA/RNA-binding proteins, which were recently recognized as a new class of DDR players. CNAs in genes/gene families, involved in MErT/EMT and DDR, were recurrent across patients and mostly segregated in the MErT/EMT category. In addition, recurrent gains of regions in chromosome 1 involving many MErT/EMT gene families and loss of one arm or the entire chromosome 6 affecting relevant immune-regulatory genes were recorded.ConclusionsThe emerging picture is an extreme inter-tumor heterogeneity, characterized by the concurrent deregulation of the DDR and MErT/EMT dynamic and plastic programs that could favour genomic instability and explain the refractory DSRCT profile.

Abstract 423: Investigating the germline deleterious rare variants for genetic susceptibility of esophagus squamous cell carcinomas by target sequencing

Abstract Purpose: The genetic basis of esophageal squamous cell carcinoma (ESCC) still remains unclear. Several genome-wide association studies identified some common variants associated with small effects. Here we aimed to identify some rare deleterious variants in cancer predisposition genes, which may contribute to the genetic susceptibility of ESCC with a large effect, using family history-positive (FH+) samples from the Mainland China Henan high-risk region by targeted sequencing. Methods: A two-phase study was conducted, the discovery phase and the validation phase. In the discovery phase, we sequenced the blood DNAs of 214 independent family history ESCC cases and 123 non-ESCC hospital controls by Illumina HiSeq platform using a custom capture kit. The capture kit included more than 1000 genes, which were cancer-related genes, DNA repair genes, and other genes reported in some ESCC functional studies. For each gene, the exonic region plus 3'-UTR, 5'-UTR, upstream and downstream regions were designed for targeted capture with a total capture size of ~11 Mb. The sequencing reads were aligned to the human reference genome (GRCh37/hg19) using burrows-wheeler aligner (bwa). PCR duplication was marked by Picards. Local realignment was performed with genome analysis toolkit (GAKT). Annovar was used to do the annotation. Loss-of-function (LoF) variants, including splicing, stopgain, and frameshift InDel variants in the cases and controls were identified. The damaging effect of the missense variants was predicted by the combination of 4 different bioinformatics tools (CADD score, MutationTaster, SIFT, Polyphen2). We also used the 1000genomes or ExAC dataset as public controls. All the identified LoF variants in BRCA2 were validated by Sanger sequencing. Results: There was a total of 110 LoF genes identified. Pathway enrichment analysis of these genes found that they were enriched in the Fanconi Anemia (FA) pathway after the background correction of all captured genes. BRCA2 is in the FA pathway and was found to have the most LoF variants; this included 6 individuals from the 214 FH+ cases, and only 1 in the control cohort. We did not find many somatic mutations in ESCC tissues in BRCA2. BRCA2 expression was also significantly upregulated in ESCC tumor tissues, as compared to adjacent normal tissues from in-house and 3 other publicly available RNA-seq datasets. The variant spectra of candidate genes were compared with the public controls to filter out some genes. Conclusions: In the discovery study, we identified some LoF variants in BRCA2 and other genes in the FA pathway that may contribute to ESCC genetic susceptibility. Further validation with a larger sample size is now underway. Acknowledgements: This work was supported by the Research Grants Council Collaborative Research Fund grant C7031-15G and the Asian Fund for Cancer Research to MLL. Citation Format: Lvwen Ning, Josephine Mun Yee Ko, Lisa Chan Lei, Li Dong Wang, Maria Li Lung. Investigating the germline deleterious rare variants for genetic susceptibility of esophagus squamous cell carcinomas by target sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 423.

Transcriptomic profiling of mitochondrial dysfunction induced apoptosis in accelerated cardiovascular disease

IntroductionVascular calcification is a significant contributor to accelerated cardiovascular disease and mortality in patients with chronic kidney disease (CKD). We previously reported that heat shock protein 70 (HSP70) induction can inhibit human aortic smooth muscle cell (HA‐SMC) calcification in CKD. In addition, our data indicated mitochondrial complexes II, IV, NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) activities were preserved by HSP70 induction leading to reduced apoptosis. Apoptosis occurs with either receptor mediated extrinsic pathway or the mitochondrial‐dependent intrinsic pathway. The molecular mechanisms of HSP70 vasculo‐protective effects involving mitochondrial dysfunction induced apoptosis are currently unknown.HypothesisThe goal of the present study was to investigate the transcriptomic profile of genes involved in mitochondrial function and vasculo‐protective mechanisms of HSPs in apoptosis induced vascular calcification.MethodsHuman arteries were collected from healthy and CKD patients. Human aortic endothelial cells (HAECs) was treated in calcification medium (CM: 5mM β‐glycerolphosphate and 5mM CaCl2) in time‐course experiments (0–48 hours), in vitro. Total RNA from human arteries and HAECs was extracted using the Nucleospin RNA isolation kit. cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). Five million single‐ended raw reads from each sample were mapped to the human reference genome consortium GRCh38 by Burrows‐Wheeler Aligner (BWA). Gene analysis were performed by the combinations of fold‐changes on log 2 ratio, and p value &lt; 0.05.ResultsIn healthy human arteries, we found that HSPA5 (fold change, FC, 3.85), HSPA1 (FC=3.64), and HSPA9 (FC=3.85) expression was preserved and Bcl2 associated Athanogene 1 (BAG1) (FC=7.44) was upregulated. However, mitochondrial fusion regulation genes MFN1 (FC=1.83) and OPA1 (FC=2.33) were down‐regulated in CKD arteries together with downregulation of downstream anti‐apoptotic gene Bcl2 (FC=4.52) and up‐regulation of pro‐apoptotic gene BAX (FC=2.77) and P53 (FC=5.35). Lastly, caspase 3 (FC=2.6) and cytochrome c (FC=2.29) expression were significantly increased in CKD arteries compared to control. In the primary HAECs, our data indicates that BAG1 was significantly increased at 6 hours prior to upregulation of Bcl2 at 12 hours in HAECs after CM treatment. BAG1 is a co‐chaperone for heat‐shock protein 70 (HSP70) that coordinates signals for cell growth in response to stress. Stress respondent genes, DNAJB6 (HSP40) and HSPA5 (HSP70 member5) was significantly increased at 24 hours and 12 hours respectively in CM‐treated HAECs.ConclusionComplex differential gene regulation involving preservation of mitochondrial function and cell apoptosis may be involved in the pathogenesis of accelerated vascular calcification. Decreased HSPs in CKD arteries may play the key role in mitochondrial dysfunction induced endothelial cells' apoptosis, which further leading to vascular calcification. Our findings may serve to inform the rational development of therapeutic strategies for vascular calcification involving therapeutic targeting of HSPs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organellar genome assembly methods and comparative analysis of horticultural plants

Although organellar genomes (including chloroplast and mitochondrial genomes) are smaller than nuclear genomes in size and gene number, organellar genomes are very important for the investigation of plant evolution and molecular ecology mechanisms. Few studies have focused on the organellar genomes of horticultural plants. Approximately 1193 chloroplast genomes and 199 mitochondrial genomes of land plants are available in the National Center for Biotechnology Information (NCBI), of which only 39 are from horticultural plants. In this paper, we report an innovative and efficient method for high-quality horticultural organellar genome assembly from next-generation sequencing (NGS) data. Sequencing reads were first assembled by Newbler, Amos, and Minimus software with default parameters. The remaining gaps were then filled through BLASTN search and PCR. The complete DNA sequence was corrected based on Illumina sequencing data using BWA (Burrows–Wheeler Alignment tool) software. The advantage of this approach is that there is no need to isolate organellar DNA from total DNA during sample preparation. Using this procedure, the complete mitochondrial and chloroplast genomes of an ornamental plant, Salix suchowensis, and a fruit tree, Ziziphus jujuba, were identified. This study shows that horticultural plants have similar mitochondrial and chloroplast sequence organization to other seed plants. Most horticultural plants demonstrate a slight bias toward A+T rich features in the mitochondrial genome. In addition, a phylogenetic analysis of 39 horticultural plants based on 15 protein-coding genes showed that some mitochondrial genes are horizontally transferred from chloroplast DNA. Our study will provide an important reference for organellar genome assembly in other horticultural plants. Furthermore, phylogenetic analysis of the organellar genomes of horticultural plants could accurately clarify the unanticipated relationships among these plants.

A Pipeline for Markers Selection Using Restriction Site Associated DNA Sequencing (RADSeq)

Motivation: The discovery and assessment genetic variants for Next Generation Sequencing (NGS), including Restriction site Associated DNA sequencing (RADSeq), is an important task in bioinformatics and comparative genetics. The genetic variants can be single-nucleotide polymorphisms (SNPs), insertions and deletions (Indels) when compared to a reference genome. Usually, the short reads are aligned to a reference genome at first using NGS alignment software, such as the Burrows- Wheeler Aligner (BWA). The alignment is usually stored into a BAM file, a binary format of standard SAM (Sequence Alignment/Map) protocol. Then analysis software, such as Genome analysis Toolkit (GATK) or SAMTools [30] [31], together with scripts written in R programming language, could provide an efficient solution for calling variants. We focused on RADSeq-based marker selection for Arabidopsis thaliana . RADSeq consists short reads that do not cover the whole reference genome. Finally, SNPs as output in Variant Call Format (VCF) have been visualized by Integrative Genomics Viewer (IGV) software. We found that the visualization of SNPs and Indels is helpful and provides us with valuable insights on marker selection. We found that applying Chi-Square test for all target genotypes, which are homozygous reference 0/0, heterozygous variants 0/1 and homozygous variants 1/1, to test Hardy-Weinberg Equilibrium (HWE) in order to reduce false positive rate significantly and we showed that our pipeline is efficient in RADSeq-based marker selection.

A Pipeline for Markers Selection Using Restriction Site Associated DNA Sequencing (Radseq)

The discovery and assessment of genetic variants for Next Generation Sequencing (NGS), including Restriction site Associated DNA sequencing (RADSeq), is an important task in bioinformatics and comparative genetics. The genetic variants can be single-nucleotide polymorphisms (SNPs), insertions and deletions (Indels) when compared to a reference genome. Usually, the short reads are aligned to a reference genome at first using NGS alignment software, such as the Burrows- Wheeler Aligner (BWA). The alignment is usually stored into a BAM file, a binary format of standard SAM (Sequence Alignment/Map) protocol. Then analysis software, such as Genome analysis Toolkit (GATK) or SAMTools, together with scripts written in R programming language, could provide an efficient solution for calling variants. In this project, we focus on RADSeq-based marker selection for Arabidopsis thaliana. RADSeq consists of short reads which do not cover the whole reference genome. In order to obtain four call-sets of SNPs as output in Variant Call Format (VCF), SNPs have been called by GATK or SAMTools. Then VCF files have been visualized by Integrative Genomics Viewer (IGV) software. We found that the visualization of SNPs and Indels has been very helpful and has provided us with valuable insights on marker selection. We found that applying Chi-Square test for all target genotypes, which are homozygous reference 0/0, heterozygous variants 0/1 and homozygous variants 1/1, to test Hardy-Weinberg Equilibrium (HWE) in order to reduce false positive rate significantly. We show that our pipeline is efficient in RADSeq-based marker selection.

P177 NGS typing results using oxford nanopore sequencing: Can minion data be reliably used for HLA typing?

Aim Nanopore sequencing is one of the latest DNA sequencing technologies, in which single molecules are sequenced directly. It offers the potential of read lengths of tens of kilobases. However, the reads are relatively error prone. Especially the length of homopolymers and repetitive sequences cannot be determined accurately. Here we present the typing results for HLA samples using Oxford Nanopore Technologies (ONT). Methods We have developed NGSengine® (GenDx), a software package to perform HLA typing based on NGS data. NGSengine uses an integrated HLA typing approach which includes determining the HLA locus, aligning reads, phasing and typing into a single method. At DKMS Life Science Lab, the MinION sequencer from ONT has been used for full-length HLA sequencing. We have used a benchmarking set of 30 samples, 10 each for HLA-A, HLA-B and HLA-C to explore 3 successive Nanopore chemistries R7.3, R9 and R9.4. Results Using the successive ONT chemistries, we have mapped the template-complement (2D) reads to the known reference alleles using Burrows-Wheeler Aligner, resulting in a mappability increasing from 98 to 99.5%. Additionally, we have used NGSengine to map the reads to the allele database, increasing the mappability from 4 to 70%. The lower mappability with NGSengine is due to the noise present within the data and the thresholds set within NGSengine. For most of the validation samples the best match presented by NGSengine corresponds with the pretyping, but all have exon mismatches in homopolymers. Noteworthy, for the samples for which the best match did not match the pretype, a Null allele was called due to the incorrect length of a homopolymer. Conclusion With the current status of MinION data quality, the results look promising but cannot yet be applied for routinge HLA typing. 1D 2 basecalling with a slight change to the chemistry (R9.5) will be available soon. We will present an update on this chemistry, and the improvements concerning homopolymers.

CRISPR-DAV: CRISPR NGS data analysis and visualization pipeline.

The simplicity and precision of CRISPR/Cas9 system has brought in a new era of gene editing. Screening for desired clones with CRISPR-mediated genomic edits in a large number of samples is made possible by next generation sequencing (NGS) due to its multiplexing. Here we present CRISPR-DAV (CRISPR Data Analysis and Visualization) pipeline to analyze the CRISPR NGS data in a high throughput manner. In the pipeline, Burrows-Wheeler Aligner and Assembly Based ReAlignment are used for small and large indel detection, and results are presented in a comprehensive set of charts and interactive alignment view. CRISPR-DAV is available at GitHub and Docker Hub repositories: https://github.com/pinetree1/crispr-dav.git and https://hub.docker.com/r/pinetree1/crispr-dav/. xuning.wang@bms.com.

Abstract 1564: A computational framework for removing mouse contamination in tumors sequenced from patient-derived xenografts

Abstract Introduction: Using patient derived xenograft (PDX) models has become an effective way for investigating response to standard or new therapeutics in cancer. Human cancer cells injected in mice are allowed to establish tumors and subjected to desired treatments. The PDX tumors are later harvested and characterized, often by massive parallel sequencing. However, a major challenge with analyzing these data is the presence of stromal mouse genomic material, frequently resulting in artifacts in downstream variant detection. We present a computational method to eliminate mouse contamination in PDX. Method: We used the Burrows-Wheeler Aligner to map reads obtained from sequencing the PDX samples to a combined human and mouse reference genome . We remove reads that are mapped to the mouse reference. The remaining reads are then processed through standard mutation calling pipelines for somatic mutation detection. To test the efficacy of our method, we created in silico mixtures of human and mouse whole-exome sequencing reads from a melanoma patient’s tumor and an immortalized mouse cell line captured with human exome baits. We then carried out a sensitivity analysis to examine how changing the mean target coverage of sequencing, or mouse contamination levels affects our results. For each of the computational experiments, we evaluated somatic mutations detected from the synthetic samples in comparison to the original human sample. Results: We calculated the sensitivity and specificity of detecting somatic mutations to determine our algorithm’s performance. In all instances, we found greater than 99% for both sensitivity and specificity. Conclusions: Our results demonstrate that our method works accurately towards removing mouse reads in PDX samples. This task could also be applied to separating sequence reads from other species. Citation Format: Ali Amin-Mansour, Judit Jané-Valbuena, Xinmeng Jasmine Mu, Levi Garraway. A computational framework for removing mouse contamination in tumors sequenced from patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1564. doi:10.1158/1538-7445.AM2017-1564

Validation of a Next-Generation Sequencing Pipeline for the Molecular Diagnosis of Multiple Inherited Cancer Predisposing Syndromes

Despite the growing knowledge of the genetic background behind the cancers that occur in a context of hereditary predisposition, personal or family cancer history may not be clear enough to support directional gene testing. Defined targeted next-generation sequencing gene panels allow identification of the causative disease mutations of multigene syndromes and differential diagnosis for syndromes with phenotypically overlapping characteristics. Herein, we established a next-generation sequencing analysis pipeline for the molecular diagnosis of multiple inherited cancer predisposing syndromes using the commercially available target sequencing panel TruSight Cancer. To establish the analysis pipeline, we included 22 control samples with deleterious mutations covering all genes currently analyzed at our institution by standard Sanger sequencing. We tested the pipeline using 51 samples from patients with a clinical diagnosis of neurofibromatosis type 1 (NF1), 10 of which without previous molecular characterization of the causative NF1 mutations. We propose a thoroughly validated analysis pipeline that combines Isaac Enrichment, Burrows-Wheeler Aligner Enrichment, and NextGENe for the alignment and variant calling, and GeneticistAssistant for variant annotation and prioritization. This pipeline allowed the identification of disease-causing mutations in all 73 patients, including a large duplication of 37 bp in NF1. We show that high sensitivity and specificity can be achieved by using multiple bioinformatic tools for alignment and variant calling and careful variant filtering, having in mind the clinical question.