Burrows-Wheeler Aligner Research Articles

Subclonal mutational signatures in breast cancer brain metastases.

e13108 Background: Breast cancer is the leading cause of cancer-related deaths among women in Brazil and the cancer type with highest incidence. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in advanced stages, survival rate is reduced to about 24%, and 90% of women in stage IV die of metastatic complications. Brain is one of the most common metastatic sites in breast cancer patients, and even though it is a site with poor prognosis, the mutational profile of brain tumors is poorly described in the literature. Therefore, we understand that the identification of this profile may contribute to elucidate mechanisms associated with tumor progression. The aim of this study was to reconstruct the subclonal evolution of breast cancer metastases in the brain. Methods: To carry out this study, the whole exome was accessed through Next Generation Sequencing (NGS) using the Illumina platform in DNA samples extracted from buffy coat cryopreserved and paraffin material of breast tumors and paired brain metastases (n=10). The median read coverage was 68x. DNA was aligned to a Human Reference Genome using the Burrows–Wheeler Aligner, Genome Analysis Toolkit was used for germline short variants calling, and Mutect2 and MuSE were used for variant calling with somatic character (SNVs and INDELs). FACETS was used for assessing the somatic DNA copy number (Copy Number Aberrations). PyClone-VI tool was used for the subclonal reconstruction and to infer the phylogeny it was considered the mutation loci, Cancer Cell Fraction (CCF) and clustering assignments, using R Statistical Environment and Stats, Argparse, and Devtools packages. Results: We identified the mutational signature in these patients with subclonal and clonal mutations that segregated in approximately two clusters with a median between ten and forty variants related with tumor progression process. Conclusions: These findings show a set of variants with driver potential for breast cancer brain metastasis, bringing new perspectives in identifying mechanisms associated with disease progression.

Forensic DNA phenotyping using Oxford Nanopore Sequencing system.

In forensic science, the demand for precision, consistency, and cost-effectiveness has driven the exploration of next-generation sequencing technologies. This study investigates the potential of Oxford Nanopore Sequencing (ONT) Technology for analyzing the HIrisPlex-S panel, a set of 41 single nucleotide polymorphism (SNP) markers used to predict eye, hair, and skin color. Using ONT sequencing, we assessed the accuracy and reliability of ONT-generated data by comparing it with conventional capillary electrophoresis (CE) in 18 samples. The Guppy v6.1 was used as a basecaller, and sample profiles were obtained using Burrows-Wheeler Aligner, Samtools, BCFtools, and Python. Comparing accuracy with CE, we found that 62% of SNPs in ONT-unligated samples were correctly genotyped, with 36% showing allele dropout, and 2% being incorrectly genotyped. In the ONT-ligated samples, 85% of SNPs were correctly genotyped, with 10% showing allele dropout, and 5% being incorrectly genotyped. Our findings indicate that ONT, particularly when combined with ligation, enhances genotyping accuracy and coverage, thereby reducing allele dropouts. However, challenges associated with the technology's error rates and the impact on genotyping accuracy are recognized. Phenotype predictions based on ONT data demonstrate varying degrees of success, with the technology showing high accuracy in several cases. Although ONT technology holds promise in forensic genetics, further optimization and quality control measures are essential to harness its full potential. This study contributes to the ongoing efforts to refine sequence read tuning and improve correction tools in the context of ONT technology's application in forensic genetics.

Next-generation DNA sequencing of Panax samples revealed new genotypes: Burrows-Wheeler Aligner, Python-based abundance and clustering analysis

BackgroundThere are two major species of the Panax genus, namely Panax ginseng and Panax quinquefolius. Other than the nucleic acid test and nucleic acid amplification test, DNA sequencing can be used to authenticate the species of ginseng samples, especially when their physical forms cannot be used for differentiation. MethodIn this work, next generation sequencing was used to obtain millions of reads from fourteen ginseng samples (root, powder, and granule). Then Gaussian Mixture clustering analysis was applied to analyze the reads from each sample. Results and DiscussionA new genotype has been revealed in this study. Two samples have been authenticated with certainty, while the others may be hybrid in nature as revealed by the clustering results.

Family analysis and literature study of hereditary hypophosphatemic rickets with hypercalciuria

BackgroundHereditary hypophosphatemia rickets with hypercalciuria (HHRH) is a rare autosomal recessive disorder characterised by reduced renal phosphate reabsorption leading to hypophosphataemia, rickets and bone pain. Here, we present a case of HHRH in a Chinese boy.Case presentationWe report a 11-year-old female proband, who was admitted to our hospital with bilateral genuvarum deformity and short stature. Computed Tomography (CT) showed kidney stones, blood tests showed hypophosphatemia, For a clear diagnosis, we employed high-throughput sequencing technology to screen for variants. Our gene sequencing approach encompassed whole exome sequencing, detection of exon and intron junction regions, and examination of a 20 bp region of adjacent introns. Flanking sequences are defined as ±50 bp upstream and downstream of the 5′ and 3′ ends of the coding region.The raw sequence data were compared to the known gene sequence data in publicly available sequence data bases using Burrows-Wheeler Aligner software (BWA, 0.7.12-r1039), and the pathogenic variant sites were annotated using Annovar. Subsequently, the suspected pathogenic variants were classified according to ACMG’s gene variation classification system. Simultaneously, unreported or clinically ambiguous pathogenic variants were predicted and annotated based on population databases. Any suspected pathogenic variants identified through this analysis were then validated using Sanger sequencing technology. At last, the proband and her affected sister carried pathogenic homozygous variant in the geneSLC34A3(exon 13, c.1402C > T; p.R468W). Their parents were both heterozygous carriers of the variant. Genetic testing revealed that the patient has anLRP5(exon 18, c.3917C > T; p.A1306V) variant of Uncertain significance, which is a rare homozygous variant.ConclusionThis case report aims to raise awareness of the presenting characteristics of HHRH. The paper describes a unique case involving variants in both theSLC34A3andLRP5genes, which are inherited in an autosomal recessive manner. This combination of gene variants has not been previously reported in the literature. It is uncertain whether the presence of these two mutated genes in the same individual will result in more severe clinical symptoms. This report shows that an accurate diagnosis is critical, and with early diagnosis and correct treatment, patients will have a better prognosis.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH‐related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3′UTR and 5′UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Genomic analysis from BLASST-1 (Bladder Cancer Signal Seeking Trial) of nivolumab, gemcitabine, and cisplatin in patients with MIBC undergoing cystectomy.

559 Background: BLASST-1 is multi-center phase II trial evaluating neoadjuvant nivolumab (N) with gemcitabine-cisplatin (GC) for patients (pts) with MIBC undergoing radical cystectomy (RC) (NCT03294304). 41 pts with MIBC (cT2-T4a, N≤1, M0) were enrolled between Feb 2018 and June 2019; (cT2N0 90%, cT3N0 7%, cT4N1 3%). Pts received C (70mg/m2) IV on D1, G (1000mg/m2) on D1, D8 and N (360 mg) IV on D8 every 21 days for 4 cycles followed by RC within 8 wks. Primary endpoint was pathologic down staging (PaR; ≤pT1N0). Safety, Relapse-free survival (RFS), Progression-free survival (PFS) and biomarker analyses were secondary endpoints. PaR rate was 65.8%, pCR (≤pT is N0) rate was 49% and there were no safety concerns or delays to RC. At a median follow-up of 15.8 mos, 12-month RFS rate was 85.4% and PFS including death from any cause was 83%. We now report correlation of PaR with tumor mutation patterns of responders (R) and non-responders (NR) using Whole Genome Sequencing (WGS). Methods: DNA was extracted from pre-treatment tumor samples and sequenced to an average depth of 150X and DNA extracted from matched normal tissue (peripheral blood) to a mean depth of 50X. We analyzed 32 Tumor/Normal pairs and 6 Tumor only Whole Genome Samples; 26 pts were classified as (R) and 12 as (NR). Raw sequencing data samples were aligned to hg38 genome build using Burrows-Wheeler Aligner (BWA) followed by duplicate-read removal and base-quality score recalibration using the Genome Analysis Toolkit (GATK). Somatic SNV and INDELS were called by combination of 5 callers (Mutect2, Strelka, VarScan, Platypus and SomaticSniper) for Tumor/Normal pairs and Mutect2 for Tumor only samples following the guidelines of GDC DNA-Seq analysis pipeline. Set of genes known to be significantly mutated in MIBC and shown to impact response/resistance to chemotherapy/immunotherapy respectively were selected. Samples were grouped by response status. Results: Median tumor mutational burden (TMB) for the dataset closely matches with TCGA results. Genes ERCC2, ARID1A, DSP, HMCN1, ERBB2, ERBB3, STAG2, BRCA2 present in (R) only, whereas VHL, ERCC4, PALB2, CDKN2A and CSMD3 present in (NR) only. We also grouped genes based on their biological processes by known Oncogenic Signaling Pathways and the 3 most common pathways are Genome Integrity, RTK Signaling and Transcription Factor with the latter two primarily enriched in (R). We identified a SNP rs2853669 in 5 (NR) and 14 (R). Conclusions: The genomic analysis suggests ERCC2, ARID1A, DSP, HMCN1, ERBB2, ERBB3,STAG2, BRCA2 correlated with PaR and VHL, ERCC4, PALB2, CDKN2A and CSMD3 correlated with lack of response. In addition, RTK Signaling and Transcription Factor were primarily enriched in (R). This genomic correlation of response/resistance to neoadjuvant chemo-immunotherapy in MIBC can help guide future biomarker-driven studies. Clinical trial information: NCT03294304 .

Prenatal diagnosis of Noonan syndrome in a set of monozygotic twins- a case report

BackgroundWe report a pair of dichorionic diamniotic (DCDA) twin pregnancy affected by Noonan syndrome (NS) with a novel mutation of LZTR1 determined by genetic analysis.Case presentationA pregnant woman with monozygotic twins (DCDA) at 12 + 2 weeks gestation was referred to our center. This was her second pregnancy following a previous delivery of a healthy infant. Nuchal translucency of two fetuses was 11.2 mm (CRL 62.0 mm) and 6.9 mm (CRL 62.1 mm) respectively. Ultrasound examination indicated cystic hygroma and hypoplastic ear. The couple was not consanguineous, and both had normal phenotype. Familial hereditary disease was also excluded. Under ultrasound guidance, 30 mg of chorionic villi was obtained for karyotyping, quantitative fluorescent polymerase chain reaction (QF-PCR), chromosomal microarray analysis(CMA), and Trio-whole-exome sequencing(WES) examination. We used the “target region capture and sequencing” for WES, and the BWA (Burrows Wheeler Aligner) Multi-Vision software package for the data analysis. The results of all these tests were normal except WES detected a c.427 A > G mutation in the exonic region of the LZTR1 gene and a p. Asn143Asp novel heterozygous mutation associated with NS in this pair of twins. In addition, WES suggested that the mutation in the twin fetuses originated from the mother. When the mother got the genetic test report, she came to our fetal medicine department for genetic counseling and she declined the appointment with a clinical geneticist. The couple opted to terminate the pregnancy. Because the patient did not choose to terminate the pregnancy at our hospital, we were unable to take further examination. With the help of colleagues in another hospital, photos of the fetuses were taken. Compared with the prenatal ultrasound results, the appearance of the “cystic hygroma” and “hypoplastic ear” was consistent with the ultrasound. The couple were depressed after knowing this pathogenic result and although we advised the mother to take further investigation, they refused.ConclusionThe mutant locus might be incompletely dominant, which led to an abnormal fetal phenotype such as cystic hygroma and hypoplastic ear.

Genome-Wide Association Study Identifies Genetic Variants Associated with Rotator Cuff Tear-A Pilot Study.

A rotator cuff is a muscle and tendon surrounding the shoulder joint, and a rotator cuff tear can be caused by overuse or injury, which leads to great pain in affected individuals. However, rotator cuff tear is a multifactorial process whose underlying mechanism is still unclear. Many previous studies have suggested an important role of genetic predisposition, such as single-nucleotide polymorphisms (SNPs), in explaining the genesis of tendinopathy. This study aimed to identify specific genes or genetic variants associated with rotator cuff tears by performing a genome-wide association study (GWAS) using an independent case of rotator cuff tears. GWAS was performed using data from CHA Bundang Medical Center with 20 cases of rotator cuff tears, and 20 cases of healthy controls genotyped on the Illumina HiSeq 2500. Tests of association were performed using the Burrows–Wheeler Aligner (BWA) software at 284,246 SNPs. Data were filtered based on sequence ontology, minor allele frequency, and Hardy–Weinberg equilibrium values, and SNPs were considered significant if the p-value was <0.05. The tests of association revealed more than 20 significantly associated SNPs. SNPs showing the highest significance occurred in candidate genes, including LAIR2 (rs2287828, OR 9.116, p-value 5.49 × 10−4) on chromosome 19 and CRIPAK (rs9328733, OR 6, p-value 1.11 × 10−3) and REST (rs2228991, OR 8.222, p-value 1.20 × 10−3) on chromosome 4. This study attempted to identify genetic variants influencing rotator cuff tears through a genome-wide association study using a dense set of SNPs. More than 20 SNPs were significantly associated with rotator cuff tears. The major limitation of this study is that it was conducted on a small study group and requires further validation. Nevertheless, the identification of potential genetic variants related to rotator cuff injury would aid in the early detection of individuals at risk for the development of tendinopathy and will provide insight into future gene therapies.

Iteratively mapping ancient DNA to reconstruct highly divergent mitochondrial genomes: An evaluation of software, parameters and bait reference

Abstract The ability to obtain ancient DNA (aDNA) from extinct species has greatly improved our ability to study their evolutionary histories. Most aDNA studies focus on mitochondrial DNA for both population‐level and species‐level insights, due to its relatively high abundance and increased obtainability. Although several approaches are available to assemble mitogenomes, high levels of DNA damage and non‐target contaminating DNA make many approaches unsuitable for aDNA. Further complicating matters for most extinct species is the lack of a sufficiently closely related extant relative to act as a reference. Iterative mapping can assemble mitogenomes without using a close reference sequence, and has been applied to aDNA from various extinct species. Despite its widespread use, the accuracy of the reconstructed assemblies is yet to be comprehensively assessed. Here, we investigated the influence of (i) mapping software (Burrows–Wheeler Alignment [BWA], MITObim and NOVOplasty), (ii) parameters and (iii) bait reference phylogenetic distance on assembly accuracy using two simulated datasets: spotted hyena and various mammalian bait references; southern cassowary and various avian bait references, and two empirical datasets: extinct cave hyena; two extinct sloth species. Specifically, we assessed the accuracy of results through three parameters: pairwise distance to a reference conspecific mitogenome, number of incorrectly inserted base pairs and total length of the assembly. We found large discrepancies in the accuracy of reconstructed assemblies using different mapping software, parameters and bait references. Results also varied depending on the dataset, thereby making broad, generalised conclusions difficult. However, the overall most accurate results were obtained with either BWA using a minimum mapping quality of 20 and relaxed mismatch parameters (‐n 0.001 ‐o 2), or MITObim using a mismatch value of 3 or 5. We also show accuracy could be further improved by combining results from multiple bait references. Our study provides information on how divergent mitogenomes are best reconstructed from aDNA. By obtaining the most accurate reconstruction possible, one can be more confident as to the reliability of downstream analyses, and the evolutionary inferences made from them.

PRKG1 mutation identified by whole-exome sequencing: a potential genetic etiology for He-Zhao deficiency

Objective: He-Zhao deficiency was originally described as a severe type of nonsyndromic hypodontia, and the causative gene locus was mapped to chromosome 10q11.2. The aim of this study was to identify potential genetic mutations that could cause He-Zhao deficiency. Methods: Patients with He-Zhao deficiency and their unaffected relatives of the large pedigree were investigated. The whole-exome sequencing using next-generation sequencing was employed to identify genetic variants. The data generated from the whole-exome sequencing using the Illumina Novaseq 6000 system were further analyzed by Burrows–Wheeler Aligner software, Sequence Alignment/Map tools and ANNOVAR tool. In vitro luciferase assay was used to investigate the effect of the detected mutation on gene expression. R environment was used to conduct t-tests. The study protocol was approved by the Research Ethics Committee of Bio-X Institutes, Shanghai Jiao Tong University (M2011004). Results: The exomes of five patients with He-Zhao deficiency and two of their unaffected relatives identified a mutation in PRKG1α as the molecular etiology of the disease. The variant c.-144 C&gt;A of PRKG1 isoform 1 cosegregated with permanent tooth agenesis in 93 family members who were older than 12, at which time the primary teeth should have been replaced with permanent teeth. Functional studies suggested that the mutant allele promotes gene transcription by increasing its promoter activity. Conclusion: c.-144 C&gt;A variant of PRKG1α involving odontoclast-associated root resorption is responsible for He-Zhao deficiency, unlike other forms of hypodontia, which typically involve odontoblast dysfunction.

Next-Generation Sequencing Highlights of Diffuse Large B-cell Lymphoma in a Tertiary Care Hospital in North India.

Introduction: Next-generation sequencing (NGS) elucidates the diffuse large B-cell lymphoma (DLBCL) genetic characteristics by finding recurrent and novel somatic mutations. This observational study attempted to create an NGS panel with a focus on identifying novel somatic mutations which could have potential clinical and therapeutic implications. This panel was created to look for mutations in 133 genes chosen on basis of a literature review and it was used to sequence the tumor DNA of 20 DLBCL patients after a centralized histopathologic review.Methods: The study included 20 patients having DLBCL. The quality and quantity of tumor cells were accessed by H&E staining and correlated with histopathology and Immunohistochemistry (IHC) status. Patients were grouped as ABC (activated B-cell), PMBL (primary mediastinal large B-cell lymphoma), and other or unclassified subtypes. The lymphoma panel of 133 was designed on targeted sequencing of multiple genes for the coding regions through NGS. The libraries were prepared and sequenced using the Illumina platform. The alignment of obtained sequences was performed using Burrows-Wheeler Aligner and identification of somatic mutations was done using LoFreq (version 2) variant caller. The mutations were annotated using an annotation pipeline (VariMAT). Previously published literature and databases were used for the annotation of clinically relevant mutations. The common variants were filtered for reporting based on the presence in various population databases (1000G, ExAC, EVS, 1000Japanese, dbSNP, UK10K, MedVarDb). A custom read-depth-based algorithm was used to determine CNV (Copy Number Variants) from targeted sequencing experiments. Rare CNVs were detected using a comparison of the test data read-depths with the matched reference dataset. Reportable mutations were prioritized and prepared based on AMP-ASCO-CAP (Association for Molecular Pathology-American Society of Clinical Oncology-College of American Pathologists), WHO guidelines, and also based on annotation metrics from OncoMD (a knowledge base of genomic alterations).Results: The informativity of the panel was 95 percent. NOTCH 1 was the most frequently mutated gene in 16.1% of patients followed by 12.9% who had ARID1A mutations. MYD88 and TP53 mutations were detected in 9.6% of the patient while 6.4% of patients had CSF3R mutations. NOTCH 1 and TP 53 are the most frequently reported gene in the middle age group (40-60). Mutation in MYD88 is reported in every age group. MYD88 (51%) is the most common mutation in ABC subtypes of DLBCL, followed by NOTCH 1 (44%) and SOCS 1 (33%) according to our findings. NOTCH 1 mutations are frequent in ABC and PMBL subtypes. Closer investigation reveals missense mutation is the most frequent mutation observed in the total cohort targeting 68.4% followed by frameshift deletion reported in 26.3%. Six novel variants have been discovered in this study.Conclusions: This study demonstrates the high yield of information in DLBCL using the NGS Lymphoma panel. Results also highlight the molecular heterogeneity of DLBCL subtypes which indicates the need for further studies to make the results of the NGS more clinically relevant.

Pediatric-Type Indolent B-Cell Lymphomas With Overlapping Clinical, Pathologic, and Genetic Features.

Pediatric-type follicular lymphoma (PTFL) and pediatric nodal marginal zone lymphoma (PNMZL) are rare pediatric-type indolent B-cell lymphomas (PedIBCL) that differ clinicopathologically from their adult counterparts. Accurate diagnosis is important to avoid overtreatment but is often challenging. The mutational landscape of PTFL is known and may aid diagnosis, but the genetic features of PNMZL are not well understood. We analyzed 21 cases of PedIBCL according to their clinicopathologic findings and classified them into PTFL (n=11), PNMZL (n=2), and “mixed type” tumors (n=8) showing ambiguous histology. We also analyzed 2 cases of adult B-cell lymphomas showing features of PedIBCL. Targeted sequencing of 121 lymphoma-related genes was performed. The median age of PedIBCL patients was 16 years (range: 3 to 47), and all but 1 PTFL patient were male. All patients presented with limited-stage disease, and only 1 relapsed. There were no significant differences in clinical features among the 3 PedIBCL groups. The most frequently mutated genes were MAP2K1, TNFRSF14, KMT2C, IRF8, and NOTCH2. The genetic features of all groups were similar to the established mutational landscape of PTFL. The 2 adult B-cell lymphomas cases also had MAP2K1, TNFRSF14, and IRF8 mutations, but the clinical features were not typical of PedIBCL. In summary, this study demonstrated that PTFL and PNMZL are similar diseases with overlapping clinical, pathologic, and genetic features; mixed type tumors can also occur. Atypical adult cases with similar histologic features were also observed. Therefore, the disease spectrum of PedIBCL may be much broader than is currently believed.

Abstract 5694: Accurate detection of microsatellite instability (MSI) in matched and unmatched clinical tumor samples using the Pillar oncoReveal MSI panel

Abstract Introduction: MSI is caused by mutations in mismatch repair (MMR) genes that result in increased insertions and deletions within simple repeats in the genome. The MSI-High (MSI-H) phenotype is prevalent in Lynch Syndrome, caused by the inheritance of MMR loss-of-function alleles as well as in 15% of all colorectal cancers. Accurate, timely detection of MSI is important for predicting the efficacy of immunotherapies that take advantage of increased expression of neoantigens in MSI-H tumors. The Pillar oncoReveal MSI Panel is a targeted NGS assay comprised of a single-tube multiplex PCR-based chemistry and companion Pillar Variant Analysis Toolkit (PiVAT) software with MSIsensor-based detection of tumor MSI status. We report the accuracy of the PiVAT MSI module in detection of tumor MSI status without the need for a matched normal comparator. Methods: 56 pairs of clinical tumor and matched normal FFPE samples were analyzed using the oncoReveal MSI Panel. MSI status was verified by a standard MSI detection method. Illumina sequencing was performed using a PE2x150 protocol. PiVAT determines MSI status using MSIsensor. Call performance on Burrows-Wheeler Alignment (BWA) BAM files was compared with PiVAT’s paired-end assembled and filtered BAM files (PBAM). In both cases coverage normalization and FDR thresholds were set to 1. In a second experiment, 88 clinical normal tissue samples were combined with the matched tumor/normal samples and analyzed using the PiVAT MSI module’s unmatched calling protocol using a synthetic pooled normal. Results: MSI was first assessed in matched tumor/normal samples using MSIsensor. Preliminary analyses revealed a greater separation of MSI scores between MSI-positive and -negative tumors when MSIsensor was run on PBAM vs BWA-generated BAM files. Due to high individual variability at MS sites, MSI detection is difficult for tumor samples with no available matched normal tissue. Hierarchical clustering demonstrated that a subset of MS sites exhibited low variability between normal samples and showed large differences between MSI-H tumor, and MS stable (MSS) tumor and/or normal samples when compared to a pooled normal baseline. Analyses based on the full target set did not accurately detect differences between MSI-H and MSS tumor or normal samples in the pooled normal calling context. When using a clustered subset of sites, MSI-detection was comparable with the matched tumor/normal context. Conclusions: We developed a robust assay for MSI detection in multiple tumor types that encompasses library generation, data analysis, and diagnosis with 97% sensitivity and 100% specificity. We also demonstrate that accurate MSI-detection is possible in the absence of matched normal tissue, using a pooled normal reference and a curated target set. Citation Format: Jordan Aldersley, Michael Liu, Lixing Qi, Ye Jiao, Akuah Kontor, Yue Ke, Zhaohui Wang. Accurate detection of microsatellite instability (MSI) in matched and unmatched clinical tumor samples using the Pillar oncoReveal MSI panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5694.

Whole genome sequencing of HER2+ metastatic breast cancer and CNA comparison between long term survivor and short-term survivor.

e13019 Background: The introduction of anti-HER2 therapies such as trastuzumab for HER2+ metastatic breast cancer (MBC) has led to significant improvements to disease progression. We and others have reported long-term durable complete response to trastuzumab in patients with HER2+ MBC. We hypothesise that genomic copy number alteration (CNA) and tumour mutational burden (TMB) may act as a prognostic measure of predicting response to trastuzumab in long-term survivors (LTS) compared to short-term survivors (STS). Using whole-genome sequencing (WGS) to detect these alterations, we aim to identify the molecular characteristics of primary and metastatic lesions in patients with metastatic HER2+ breast cancer. Methods: WGS was performed on samples from two patients from a HER2+ MBC cohort; a of long-term survivor (PFS 27 months; OS &gt; 60 months) and a short-term survivor (PFS 4 months; OS &lt; 14 months). Primary tumours, adjacent normal tissue and metastases were sequenced at a mean depth of 60X. Reads were aligned on the hg38 reference genome using Burrows-Wheeler Aligner (bwa), CNA were detected using Control-FREEC to evaluate total CNA burden. Single nucleotide variants were detected using GATK Mutect2 to calculate TMB. Results: Analysis of DNA chromosome disruption (fraction of the genome amplified/deleted) revealed a higher overall fraction of disrupted genome in the LTS primary tumour and metastasis compared with the STS (0.061 and 0.064 vs 0.031 and 0.047, respectively). Further delineation of the distribution of CNA burden in all genomes identified chr1 as the most altered in the STS samples whereas chr17 was the most disrupted in the LTS samples. HER2 and CDK12 were amplified in both LTS and STS samples (4 copies in LTS primary tumour and metastasis, 18 and 14 copies in STS primary tumour and metastasis respectively). A high TMB was estimated in both samples but was higher in the STS primary tumour than in the LTS one (161 vs 142, respectively). TMB was increased in LTS metastatic lesions. NRAS mutation was found in LTS primary tumour but was lost in the metastasis. Conclusions: This pilot study highlights the potential for whole-genome sequencing to identify CNA and TMB in HER2+ MBC in patients who display long-term and short-term survival. Further samples from this cohort need to be studied to investigate the relationship between CNA burden and HER2+ MBC patient survival.

Integrative Comparison of Burrows-Wheeler Transform-Based Mapping Algorithm with de Bruijn Graph for Identification of Lung/Liver Cancer-Specific Gene.

Cancers of the lung and liver are the top 10 leading causes of cancer death worldwide. Thus, it is essential to identify the genes specifically expressed in these two cancer types to develop new therapeutics. Although many messenger RNA (mRNA) sequencing data related to these cancer cells are available due to the advancement of next-generation sequencing (NGS) technologies, optimized data processing methods need to be developed to identify the novel cancer-specific genes. Here, we conducted an analytical comparison between Bowtie2, a Burrows-Wheeler transform-based alignment tool, and Kallisto, which adopts pseudo alignment based on a transcriptome de Bruijn graph using mRNA sequencing data on normal cells and lung/liver cancer tissues. Before using cancer data, simulated mRNA sequencing reads were generated, and the high Transcripts Per Million (TPM) values were compared. mRNA sequencing reads data on lung/liver cancer cells were also extracted and quantified. While Kallisto could directly give the output in TPM values, Bowtie2 provided the counts. Thus, TPM values were calculated by processing the Sequence Alignment Map (SAM) file in R using package Rsubread and subsequently in python. The analysis of the simulated sequencing data revealed that Kallisto could detect more transcripts and had a higher overlap over Bowtie2. The evaluation of these two data processing methods using the known lung cancer biomarkers concludes that in standard settings without any dedicated quality control, Kallisto is more effective at producing faster and more accurate results than Bowtie2. Such conclusions were also drawn and confirmed with the known biomarkers specific to liver cancer.

Association of intra-tumoral microbiome and response to immune checkpoint inhibitors (ICIs) in patients with metastatic renal cell carcinoma (mRCC).

372 Background: Multiple studies have suggested that the gut microbiome plays a modulatory role in ICI activity and that specific bacteria and/or cumulative microbial diversity may drive response in patients (pts) with mRCC (Routy et al Science 2018; Salgia et al Eur Urol 2020). Even though the tumor microenvironment has substantial bacterial proliferation (Heymann et al Cancer L. 2021), there is a paucity of data assessing the impact of intra-tumoral microbiota in response to ICI therapy. In this study, we sought to explore this association in pts with mRCC. Methods: Pts diagnosed with mRCC who had available RNA sequencing (RNA-seq) data collected in the course of routine clinical care and who were treated with ICIs were retrospectively identified.Intra-tumoral microbiome analysis was performed on formalin-fixed paraffin-embedded samples. Following quality and adapter trimming, RNA-seq reads were mapped to a human genome to filter host reads using the Burrows-Wheeler alignment (BWA) tool. Taxonomic classification was performed using Kraken2 and the absolute abundances of species were estimated using Bracken. The relative abundances among all non-human species were calculated. Statistical testing with Student’s t-test was performed to compare the relative abundance for all species seen within pts who responded to ICIs and those who did not. Results: Among the 28 pts (22:6, M:F) included in this analysis, 24 (86%) had clear cell histology and 20 (71%) were IMDC intermediate/poor risk. All of the samples were collected prior to starting treatment with ICIs and the majority of these (57%) were collected from the primary site. 11 pts (39%) received ICIs as first line treatment and 17 (61%) as second line. Clinical response was seen in 50% of pts included in the study and the most common rendered treatment was nivolumab (17 pts). In the overall cohort, Cutibacterium acne, Moraxella osloensis, and Pasteurella multocida had the highest relative abundances. Additionally, significant differences in relative abundances of specific bacteria were found between ICI responders and non-responders. Among these, Stenotrophomonas maltophilia (p = 0.037) and Corynebacterium sp. zg-917 (p = 0.035) had significantly higher relative abundances in pts who responded to ICIs. Conclusions: This is the first study evaluating the association between intra-tumoral microbiome and response to ICIs in pts with mRCC. Among bacteria associated with response, several have particular relevance – for instance, Corynebacterium spp. have been studied for decades as a possible adjunct to immunotherapeutic agents such as BCG. Efforts are ongoing to validate these findings in a larger cohort.

Abstract P3-09-14: Whole exome sequencing of matched primary and metastatic triple-negative breast cancer samples

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype with distinct biological features and clinical behavior. TNBC is associated with an increased risk of metastasis and recurrence. In this whole exome study, we investigated the genetic profiles of primary TNBC tumors and paired metastases using next-generation sequencing (NGS). Methods: Genomic DNA extracted from 35 paraffin-embedded formalin-fixed (FFPE) tissues (15 primary tumors that did not metastasize, 11 primary tumors that metastasized, and nine paired metastatic tumors to the lymph nodes) was analyzed by whole exome sequencing (WES). Reads were trimmed using Trim-Galore and were aligned to the reference genome GRCh38 using Burrows-Wheeler Aligner (BWA). BAM files were preprocessed to optimize variant calling, and variant calling was carried out using the GATK pipeline. Variants were annotated using SnpEff. Tumors were analyzed for single nucleotide variants (SNV), point mutations, and insertions/deletions (indels). Results: Primary tumors that did not metastasize had a higher number of variants (~13,500) than primary tumors that metastasized (~12,900). However, the number of variants was similar between the primary tumors (~12,900 variants) and their matched metastases (~12,500 variants). MUC3A was the top mutated gene both in primary tumors that did not metastasize and in those that metastasized. MUC3A was also the top mutated gene in matched metastatic lesions. Moreover, we compared the mutational status of the most frequently mutated genes in TNBC samples from the TCGA and METABRIC datasets. We found that in our dataset, TP53, MAP3K1, and PTEN were mutated in 60%, 93%, and 7% of primary TNBC tumors without metastases and in 36%, 0%, and 36% of primary TNBC tumors that metastasized. Mutations in TP53 and PTEN were found in 2/9 and 1/9 of primary and metastases pairs, respectively. No mutations in PI3KCA were observed in any of the primary or matched metastatic tumors. Conclusions: We used WES to compare the genomic landscapes of primary TNBC tumors and matched metastatic tumors, as well as of primary TNBC tumors that metastasized and those that did not metastasize. We found very similar genomic alterations between the primary and paired metastatic tumors, indicating that genomic features may be retained during metastasis. Primary tumors that did not metastasize showed a greater extent of genomic alterations than primary tumors that metastasized. Citation Format: Jaspreet Kaur, Darshan S. Chandrashekar, Zsuzsanna Varga, Emiel Janssen, Khanjan Gandhi, Karuna Mittal, Umay Kiraz, Sooryanarayana Varambally, Ritu Aneja. Whole exome sequencing of matched primary and metastatic triple-negative breast cancer samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-14.

Detecting Bacterial-Human Lateral Gene Transfer in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a very common and mostly incurable B-cell malignancy. Recent studies revealed high interpatient mutational heterogeneity and worsened therapy response and survival of patients with complex genomic aberrations. In line with this, a better understanding of the underlying mechanisms of specific genetic aberrations would reveal new prognostic factors and possible therapeutic targets. It is known that chromosomal rearrangements including DNA insertions often play a role during carcinogenesis. Recently it was reported that bacteria (microbiome)–human lateral gene transfer occurs in somatic cells and is enriched in cancer samples. To further investigate this mechanism in CLL, we analyzed paired-end RNA sequencing data of 45 CLL patients and 9 healthy donors, in which we particularly searched for bacterial DNA integrations into the human somatic genome. Applying the Burrows–Wheeler aligner (BWA) first on a human genome and then on bacterial genome references, we differentiated between sequencing reads mapping to the human genome, to the microbiome or to bacterial integrations into the human genome. Our results indicate that CLL samples featured bacterial DNA integrations more frequently (approx. two-fold) compared to normal samples, which corroborates the latest findings in other cancer entities. Moreover, we determined common integration sites and recurrent integrated bacterial transcripts. Finally, we investigated the contribution of bacterial integrations to oncogenesis and disease progression.

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

Genome-wide sequencing as a first-tier screening test for short tandem repeat expansions

BackgroundScreening for short tandem repeat (STR) expansions in next-generation sequencing data can enable diagnosis, optimal clinical management/treatment, and accurate genetic counseling of patients with repeat expansion disorders. We aimed to develop an efficient computational workflow for reliable detection of STR expansions in next-generation sequencing data and demonstrate its clinical utility.MethodsWe characterized the performance of eight STR analysis methods (lobSTR, HipSTR, RepeatSeq, ExpansionHunter, TREDPARSE, GangSTR, STRetch, and exSTRa) on next-generation sequencing datasets of samples with known disease-causing full-mutation STR expansions and genomes simulated to harbor repeat expansions at selected loci and optimized their sensitivity. We then used a machine learning decision tree classifier to identify an optimal combination of methods for full-mutation detection. In Burrows-Wheeler Aligner (BWA)-aligned genomes, the ensemble approach of using ExpansionHunter, STRetch, and exSTRa performed the best (precision = 82%, recall = 100%, F1-score = 90%). We applied this pipeline to screen 301 families of children with suspected genetic disorders.ResultsWe identified 10 individuals with full-mutations in the AR, ATXN1, ATXN8, DMPK, FXN, or HTT disease STR locus in the analyzed families. Additional candidates identified in our analysis include two probands with borderline ATXN2 expansions between the established repeat size range for reduced-penetrance and full-penetrance full-mutation and seven individuals with FMR1 CGG repeats in the intermediate/premutation repeat size range. In 67 probands with a prior negative clinical PCR test for the FMR1, FXN, or DMPK disease STR locus, or the spinocerebellar ataxia disease STR panel, our pipeline did not falsely identify aberrant expansion. We performed clinical PCR tests on seven (out of 10) full-mutation samples identified by our pipeline and confirmed the expansion status in all, showing absolute concordance between our bioinformatics and molecular findings.ConclusionsWe have successfully demonstrated the application of a well-optimized bioinformatics pipeline that promotes the utility of genome-wide sequencing as a first-tier screening test to detect expansions of known disease STRs. Interrogating clinical next-generation sequencing data for pathogenic STR expansions using our ensemble pipeline can improve diagnostic yield and enhance clinical outcomes for patients with repeat expansion disorders.