Indel Detection Research Articles

BSCI-19. EFFECT OF STEREOTACTIC RADIOSURGERY ON NON-SMALL CELL LUNG CANCER BRAIN METASTASIS: CORRELATIVE RADIOBIOLOGIC ANALYSIS FROM PHASE-II CLINICAL TRIAL NCT03398694

BACKGROUND: Stereotactic radiosurgery (SRS) is an increasingly common modality used with or without surgery for brain metastases (BM). However, biological effect of SRS to tumors in vivois not known. METHODS: Patients were treated with SRS prior to surgery as per the clinical trial protocol. The resected tumor was divided into two groups: ‘center’ and ‘periphery’ with respect to the center of SRS treatment with periphery within 50%-isodose-line. Tissue was analyzed by whole exome sequencing (WES) and compared between the two groups as well as to non-radiated control tissues. RESULTS: All sequenced samples contained greater than 95% clean reads with an average read density of 100 base pairs and mapping efficiency of >99%. Preliminary analysis focused on SNP and Indel detection. In pooled groups with n=7 replicates there was no statistically significant differences in total mutation burden in either SNP’s or Indels compared between controls and both treatment locations. Delving deeper intronic, frameshift, missense, and nonsense mutations all also showed insignificant changes between controls and center or peripheral tumor locations (p >0.5, p >0.1, p >0.4, p >0.3 respectively) hinting that at a pooled biological level there are not significant mutational burdens between treatment locations. However, at the individual level, matched comparison of SRS samples originating from the center or periphery of the same tumor showed total mutational burden differences. 6 out of 7 (86%) patients showed decreased total number of indels in peripheral vs. center and 5 out of 7 (71%) patients showed decreased number of SNP’s in peripheral vs. center locations. CONCLUSION: Ultimately, these data demonstrate the power of matched patient controls over bulk analysis in order to elucidate smaller but possibly biologically meaningful effects, and point to a possible location dependency in treatment associated mutation burden within a single patient and single tumor that may be masked at a population level.

Abstract 1381: Technical validation of Roche AVENIO sequencing platform for liquid biopsies

Abstract Purpose Circulating cell-free tumor DNA (ctDNA) is rapidly gaining attention as a valuable biosource for the detection and diagnosis of multiple types of cancer. ctDNA can be easily obtained through a minimally invasive liquid biopsy. Multiple NGS-based methods have been developed specifically for application in liquid biopsies. Before such methods can be implemented in clinical care, they need to be technically validated through measurement of independent reference standards. In the current project we technically validated the Roche AVENIO ctDNA Assay, which is based on the CAPP-seq technology [Newman AM, et al. Nat Biotechnol. 2016;34:547-55]. Its Targeted Kit interrogates 17 genes for Single Nucleotide Variants (SNVs), Insertions and Deletions (InDels), Copy Number Variations (CNVs) and gene fusions in a single workflow. Methods The detection of SNVs, CNVs, Indels and gene fusions was evaluated as follows: - SeraCare Seraseq ctDNA Complete was analyzed in four replicates at 2.5%, 0.5% and 0.1% mutant allele frequency (AF), and at 50ng and 10ng input. This material contains SNVs, InDels, CNVs and gene fusions. - An in-house characterized reference pool of patient plasma, containing SNVs and InDels at various AF (0.07% - 5.51%), was analyzed in four replicates at 50ng and 10ng input. - Plasma and serum samples obtained from patients with tissue confirmed presence of CNV were used to validate the robustness of CNV detection. Twelve samples were selected to represent a range of background DNA (serum had 2-4 times higher wildtype DNA levels compared to plasma), amplification levels (CNV levels 1.4-fold to 4.2-fold by ddPCR) and total input (10-50ng cfDNA input). - Plasma obtained from five patients with EML4-ALK gene fusion confirmed on tissue was used to further validate fusion detection. Sequencing was performed on an Illumina NextSeq on High Output mode with sixteen samples per run (20-33M read pairs per sample). Results Sensitivity for detection of SNVs and InDels was 100% for all variants down to 0.18% AF when using 50ng input, and 50% for variants at 0.10% AF. For 10ng input the variants were detected down to 0.60% AF with sensitivity of 92%. For variants with AF between 0.07% and 0.53%, sensitivity was 67%. Results from twelve plasma and serum samples obtained from patients with EGFR CNV were 100% concordant with ddPCR results. These results were confirmed in SeraCare samples at 10ng and 50ng input. Sensitivity of fusion detection in SeraCare material was 100% for AF of 0.5% and 50ng input, and 38% for input of 10ng. No fusions were detected at 50ng or 10ng input for AF of 0.1%. We furthermore confirmed 3 out of the 5 fusions found in tissue. Conclusions We have determined the lower limits of detection for SNVs, CNVs and gene fusions. Sensitivity was dependent on DNA input with performance optimized at 50ng. Our results confirm the feasibility of in-house use of the AVENIO ctDNA Targeted Kit for broad molecular profiling in liquid biopsies. Citation Format: Daan C. Vessies, Theodora C. Linders, Kalpana L. Ramkisoensing, Petra M. Nederlof, Gerrit A. Meijer, Kim Monkhorst, Daan van den Broek. Technical validation of Roche AVENIO sequencing platform for liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1381.

Abstract 2238: Characterization of T cell clonality and tumor mutation burden utilizing the TCRSeq NGS and the comprehensive NGS assays

Abstract Background: Somatic mutations are the basis for the generation of potential neoantigens recognized by antitumor T lymphocytes, which may contribute to increase T cell diversity [1]. Next-generation sequencing of expressed T cell receptor (TCR) transcripts allows for quantification and assessing clonality of T cell diversity [2]. Tumors that exhibit a low TCR diversity and potentially having a high clonality have been shown to be lower responders to immunotherapy treatments. Methods: Utilizing the Shannon entropy to arrive at a clonality readout, we sought to gain mechanistic insights and demonstrate a link between clonality and total mutation load. We conducted the analysis using a diverse set of solid tumors and hematological malignancies across three different specimen types (bone marrow (BM), peripheral blood (PB), formalin-fixed paraffin embedded (FFPE tissue)). Specifically, we used the beta TCRSeq NGS and the Comprehensive NGS assays (internally developed) to characterize sample clonality and mutation load respectively. Results: The TCRSeq NGS assay sequences the V(D)J domains of the variable region of the TCR heavy chain (involved in epitope recognition), while the hybrid capture Comprehensive NGS assay detects variants across a 236 gene panel optimized for SNV and INDEL detection. Initial analysis across the different specimen types resulted in average clonalities of BM = 0.255, PB = 0.149, FFPE = 0.092, with average tumor mutation loads of 7.6, 7.6, and 21 respectively. Categorization of all samples by increasing clonality, independent of specimen type, established a clear differentiator with samples harboring a clonality of less than 0.10 to exhibit an average mutation load of 26.33. This was statistically significant from samples harboring clonalities between 0.10 and 0.19, which had an average mutation load of 7.86, and samples with clonalities equal to or greater than 0.20, having an average mutation load of 7.67. Conclusions: Based on two different categorical analyses, lower T cell clonality readouts appear to be associated with solid tumors harboring greater genetic diversity/neoantigen burden than hematological malignancies with higher clonalities. As tumor mutation burden has been correlated with clinical outcomes of varying efficacy, this study provides further insight into the utility of T cell clonality as a “molecular tag” for immune response and resistance in oncological therapeutics. Citation Format: Hua C. Gong, Hua Gong, Andreas Papoutsis, Wen Wu, Christian Laing. Characterization of T cell clonality and tumor mutation burden utilizing the TCRSeq NGS and the comprehensive NGS assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2238.

Abstract 1346: Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay

Abstract Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina’s TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina’s TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in >500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina’s analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (> 20), and greater than 85% of MSI-low tumors characterized as TMB-low (<20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients. Citation Format: Stephanie B. Hastings, Gunjan Hariani, Victor J. Weigman, Tingting Jiang, Chen Zhao, Rajiv Raja, Traci Pawlowski, Philip Brohawn, Patrick Hurban. Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1346.

Abstract 4252: Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing

Abstract Background Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. MSI-High (MSI-H) CRC patients have been shown to respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone. Methods Our workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status. Results The above algorithm was evaluated on a cohort of 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. A second cohort evaluated includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation. Conclusions Here we present an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal. Citation Format: Amrita Pati, Hao Wang, Hamid Mirebrahim, Seng Saelee, Joshua Lefkowitz, Sean Chien, Ashla Singh, Fergal Casey, Vera Rapoport, Xiaoju Max Ma, John Lee, Alex Lovejoy, Daniel Klass, Hans-Peter Adams. Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4252.

Indel detection from Whole Genome Sequencing data and association with lipid metabolism in pigs.

The selection in commercial swine breeds for meat-production efficiency has been increasing among the past decades, reducing the intramuscular fat content, which has changed the sensorial and technological properties of pork. Through processes of natural adaptation and selective breeding, the accumulation of mutations has driven the genetic divergence between pig breeds. The most common and well-studied mutations are single-nucleotide polymorphisms (SNPs). However, insertions and deletions (indels) usually represents a fifth part of the detected mutations and should also be considered for animal breeding. In the present study, three different programs (Dindel, SAMtools mpileup, and GATK) were used to detect indels from Whole Genome Sequencing data of Iberian boars and Landrace sows. A total of 1,928,746 indels were found in common with the three programs. The VEP tool predicted that 1,289 indels may have a high impact on protein sequence and function. Ten indels inside genes related with lipid metabolism were genotyped in pigs from three different backcrosses with Iberian origin, obtaining different allelic frequencies on each backcross. Genome-Wide Association Studies performed in the Longissimus dorsi muscle found an association between an indel located in the C1q and TNF related 12 (C1QTNF12) gene and the amount of eicosadienoic acid (C20:2(n-6)).

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts

BackgroundCRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.ResultsIn this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.ConclusionsFACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

Performance assessment of variant calling pipelines using human whole exome sequencing and simulated data

BackgroundWhole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length.ResultsWe assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB.ConclusionIn this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.

PS1008 A NEW NEXT‐GENERATION SEQUENCING STRATEGY FOR THE SIMULTANEOUS DETECTION OF GENE MUTATIONS AND COPY NUMBER VARIATIONS IN MYELOID MALIGNANCIES

Background:The diagnosis of myeloid malignances includes multiple testing strategies for genetic abnormalities assessment, including gene mutations and copy number variations (CNVs), which is critical for therapeutic decision making. Although next‐generation sequencing (NGS) has been implemented in clinical diagnostic laboratories, it has focused on the detection of single nucleotide variants and small indels, while CNV detection still relies on karyotyping and fluorescence in situ hybridization (FISH).Aims:In the present study, a new strategy was developed and validated to simultaneously detect CNVs and somatic mutations in a series of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) by using a capture‐based NGS custom panel.Methods:We designed a NGS target‐DNA panel associated to a custom bioinformatic pipeline (Sophia Genetics) for one‐step identification of somatic and germline mutations in 48 genes, as well as CNV. The CNV algorithm was based on the coverage levels across samples within the same batch. We studied the complete CDS or targeted exons of 16 genes sited in chromosome regions 5q14‐q35 (5 genes), 7q22‐q36 (3 genes), 8q12‐q24 (4 genes), 20q11‐q13 (3 genes), 17p13 (1 gene, TP53), as well as other genes in 5p, 7p, 20p and 17q as control regions. We analyzed 45 diagnostic samples of myeloid malignances (39 AML and 6 MDS), that had previously been characterized by FISH and/or karyotyping, as well as by conventional molecular techniques (FLT3‐ITD, NPM1 and CEBPA mutations). Paired‐end sequencing runs were performed on a MiSeq (Illumina) genome sequencer.Results:We detected 133 pathogenic mutations in 42 patients, including all 8 FLT3‐ITD (size 18–204 bp) and 4 mutations in CEBPA. We were also able to detect exon‐level duplications, such as KMT2A (MLL) partial tandem duplication, in 3 cases. Four different CNV types were distinguished by the algorithm: del(5q), del(7q), del(20q) and trisomy 8, showing a full concordance with those alterations identified by FISH probes. All 8 patients with 5q33 deletions (8/8, 100%) were detected by NGS, as well as 5 cases with 7q35 deletion (5/5, 100%), 3 patients with trisomy 8 (3/3, 100%) and 3 with 20q12 deletion (3/3, 100%). None of the patients presented 17p abnormalities by any technique. Moreover, we detected alterations in the copy number of KMT2A gene in 4 of 5 patients with 11q23 alterations (translocation or amplification) identified by FISH and cytogenetics. One case of t(11;19)(q23;p13) was not detected by NGS, which is explained because the breakpoint region of this translocation was not covered by our design. Next panel will overcome this caveat by including the complete CDS of the KMT2A gene. Finally, NGS estimated abnormal copy numbers in three cases of 7q deletions and one trisomy 8, where FISH and cytogenetics had failed to detect any change.Summary/Conclusion:Our study demonstrates that both somatic mutations and targeted CNVs can be accurately detected by NGS covering adequate exons on selected genes. Therefore, this strategy complements conventional methods to identify biomarkers for the diagnosis, prognosis and therapy of myeloid malignances.

Copy number variant and runs of homozygosity detection by microarrays enabled more precise molecular diagnoses in 11,020 clinical exome cases

BackgroundExome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES.MethodsIn a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array.ResultsThe QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities.ConclusionsOur clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

A new massively parallel nanoball sequencing platform for whole exome research

BackgroundWhole exome sequencing (WES) has been widely used in human genetics research. BGISEQ-500 is a recently established next-generation sequencing platform. However, the performance of BGISEQ-500 on WES is not well studied. In this study, we evaluated the performance of BGISEQ-500 on WES by side-to-side comparison with Hiseq4000, on well-characterized human sample NA12878.ResultsBGISEQ demonstrated similarly high reproducibility as Hiseq for variation detection. Also, the SNVs from BGISEQ data is highly consistent with Hiseq results (concordance 96.5%~ 97%). Variation detection accuracy was subsequently evaluated with data from the genome in a bottle project as the benchmark. Both platforms showed similar sensitivity and precision in SNV detection. While in indel detection, BGISEQ showed slightly higher sensitivity and lower precision. The impact of sequence depth and read length on variation detection accuracy was further analyzed, and showed that variation detection sensitivity still increasing when the sequence depth is larger than 100x, and the impact of read length is minor when using 100x data.ConclusionsThis study suggested that BGISEQ-500 is a qualified sequencing platform for WES.

An integrated package for bisulfite DNA methylation data analysis with Indel-sensitive mapping

BackgroundDNA methylation plays crucial roles in most eukaryotic organisms. Bisulfite sequencing (BS-Seq) is a sequencing approach that provides quantitative cytosine methylation levels in genome-wide scope and single-base resolution. However, genomic variations such as insertions and deletions (indels) affect methylation calling, and the alignment of reads near/across indels becomes inaccurate in the presence of polymorphisms. Hence, the simultaneous detection of DNA methylation and indels is important for exploring the mechanisms of functional regulation in organisms.ResultsThese problems motivated us to develop the algorithm BatMeth2, which can align BS reads with high accuracy while allowing for variable-length indels with respect to the reference genome. The results from simulated and real bisulfite DNA methylation data demonstrated that our proposed method increases alignment accuracy. Additionally, BatMeth2 can calculate the methylation levels of individual loci, genomic regions or functional regions such as genes/transposable elements. Additional programs were also developed to provide methylation data annotation, visualization, and differentially methylated cytosine/region (DMC/DMR) detection. The whole package provides new tools and will benefit bisulfite data analysis.ConclusionBatMeth2 improves DNA methylation calling, particularly for regions close to indels. It is an autorun package and easy to use. In addition, a DNA methylation visualization program and a differential analysis program are provided in BatMeth2. We believe that BatMeth2 will facilitate the study of the mechanisms of DNA methylation in development and disease. BatMeth2 is an open source software program and is available on GitHub (https://github.com/GuoliangLi-HZAU/BatMeth2/).

Improved indel detection in DNA and RNA via realignment with ABRA2.

MotivationGenomic variant detection from next-generation sequencing has become established as an extremely important component of research and clinical diagnoses in both cancer and Mendelian disorders. Insertions and deletions (indels) are a common source of variation and can frequently impact functionality, thus making their detection vitally important. While substantial effort has gone into detecting indels from DNA, there is still opportunity for improvement. Further, detection of indels from RNA-Seq data has largely been an afterthought and offers another critical area for variant detection.ResultsWe present here ABRA2, a redesign of the original ABRA implementation that offers support for realignment of both RNA and DNA short reads. The process results in improved accuracy and scalability including support for human whole genomes. Results demonstrate substantial improvement in indel detection for a variety of data types, including those that were not previously supported by ABRA. Further, ABRA2 results in broad improvements to variant calling accuracy across a wide range of post-processing workflows including whole genomes, targeted exomes and transcriptome sequencing.Availability and implementationABRA2 is implemented in a combination of Java and C/C++ and is freely available to all from: https://github.com/mozack/abra2.Supplementary information Supplementary data are available at Bioinformatics online.

Fast and Quantitative Identification of Ex Vivo Precise Genome Targeting-Induced Indel Events by IDAA.

Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short "sample-to-data" turnaround time of less than 5h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.

Improved indel detection in DNA and RNA via realignment with ABRA2

Improved indel detection in DNA and RNA via realignment with ABRA2

Duplex Sequencing Accurately Detects Variants below 1/100,000 in Genes Recurrently Mutated in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a challenging disease to treat: most patients achieve remission after induction chemotherapy, but the majority eventually relapse. Minimal residual disease (MRD) after initial treatment is the best predictor of relapse and is thus a critical metric around which to develop new treatments. However, conventional MRD diagnostics, including cytology and flow cytometry, are of variable sensitivity and often only perform well in specialty centers, and there is no gold standard. Molecular tests developed to measure trace MRD in other hematological malignancies (i.e. CML) are high-resolution but assay a single, universally-present mutation, while many different genetic drivers exist in AML and these are spread among dozens of genes. Virtually every AML patient harbors a unique combination of mutations, making it difficult to design an effective universal assay. As such, most reported molecular AML MRD assays are either sensitive for mutations that are only found in a narrow subset of patients, or can screen many potential sites of mutation, but with low sensitivity.Here we present a broadly applicable Duplex Sequencing-based AML MRD assay that can readily detect mutant allele frequencies (MAF) below 1/10,000 across a large panel of genes, and below 1/100,000 in a focused panel. Conventional next generation sequencing (NGS) introduces errors during amplification and sequencing, creating a background of artifactual noise that obscures true mutations present below ~1%. Duplex Sequencing improves accuracy >100,000-fold through a molecular tagging approach whereby both strands of each original DNA duplex are ligated with a unique molecular barcode and amplified such that the reads generated from each strand can be related back to their unique original duplex. Reads can also be distinguished from those of their mate strand, thus the two strands of each DNA duplex can be compared and any discrepant nucleotide positions are discounted as errors.Our complete AML panel targets 151 exons or hotspot codons in 29 genes with a 59 kilobase (kb) hybrid-capture footprint. This region comprises loci containing single-nucleotide and short indel mutations found in approximately 90% of adult AMLs. A mean Duplex error-corrected sequence depth of 10,837 and a maximum Duplex depth of 14,967 was obtained across these targets from a single library preparation using 250 ng of sheared leukocyte DNA (Fig. 1). Duplex depth can be readily increased by preparing additional Duplex libraries from the same source DNA to achieve proportionally higher sensitivity for rarer variants. This stands in contrast to conventional NGS where, beyond a modest level, an increase in depth simply increases the number of background errors identified (Fig. 2A).We simulated low-level residual disease by mixing control DNA from a healthy young blood donor with DNA from 9 human cell lines harboring known AML mutations at dilutions from 1:100 to 1:100,000 (Table 2). The genomic loci of these 9 mutations in NRAS, KRAS and TP53 were captured with a small 1 kb probe panel. This mixture was sequenced to a mean Duplex depth of >1,000,000-fold, with the highest and lowest MAFs shown in Fig. 2B. All were close to expected frequencies (r2=0.96) with MAF as low as 6x10-6 (Fig. 3). As proof of specificity, we examined all coding nucleotide positions (excluding the 9 expected variants) and identified only 241 background variant counts out of 414,452,402 total Duplex BP, for an aggregate mutation frequency of 5.8x10-7, consistent with the estimated background of normal human aging.Our Duplex Sequencing-based AML MRD assay is flexible, broadly applicable and extremely sensitive. The assay is easily implemented using standard NGS equipment and automated cloud-based analysis software. The ~90% of AML patients served by this SNV-focused panel can be expanded to nearly 100% with complementary indel detection via targeted NGS RT-PCR. Optionally, when a patient's mutation profile from time-of-diagnosis is known, MRD testing can focus exclusively on those targets using a subset of pre-validated probes to reduce sequencing cost. Improved MRD testing will facilitate accurate prognostication, better selection among treatment options, and could serve as a surrogate endpoint in clinical trials to bring new treatments to patients faster. We are currently evaluating Duplex Sequencing MRD tests in both retrospective and prospective clinical trials. [Display omitted] DisclosuresHiggins:TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Buckley:CTI Biopharma: Employment; TwinStrand Biosciences: Consultancy. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Employment, Equity Ownership.

Validation of a Long-Read PCR Assay for Sensitive Detection and Sizing of C9orf72 Hexanucleotide Repeat Expansions

A hexanucleotide GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. Accurate determination and quantitation of the repeat length is critical in both clinical and research settings. However, because of the complexity of the C9orf72 expansion with high GC content, large size of repeats, and high rate of insertions/deletions (indels) and sequence variations in the flanking regions, molecular genetic analysis of the locus is challenging. To improve the performance characteristics for clinical testing, we evaluated a commercially available long-read C9orf72 PCR assay for research use only, AmplideX PCR/CE C9orf72 assay (AmplideX-C9), and compared its performance with our existing laboratory-developed C9orf72 expansion procedure. Overall, in comparison to the laboratory-developed C9orf72 expansion procedure, AmplideX-C9 demonstrated a more efficient workflow, greater PCR efficiency for sizing of repeat expansions, and improved peak amplitude with lower DNA input and higher analytic sensitivity. This, in turn, permitted detection of indels in the 3' downstream of the repeat expansion region in expanded alleles, showed a higher success rate with formalin-fixed, paraffin-embedded tissue specimens, and facilitated the assessment of repeat mosaicism. In summary, AmplideX-C9 will not only help to improve clinical testing for C9orf72-associated amyotrophic lateral sclerosis and frontotemporal degeneration but will also be a valuable research tool to better characterize the complexity of expansions and study the effects of indels/sequence variations in the flanking region.

Synthego's Engineered Cells Allow Scientists to "Cut Out" CRISPR Optimization [SPONSORED

Synthego's Engineered Cells Allow Scientists to "Cut Out" CRISPR Optimization [SPONSORED

High-throughput sequencing of murine immunoglobulin heavy chain repertoires using single side unique molecular identifiers on an Ion Torrent PGM

With the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT's underlying algorithms efficiently detect 98% of the introduced indels. Undetected indels are either located at the end of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. The complementary determining regions 3 (CDR3s) are returned correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers result in highly accurate HTS data if combined with the presented processing strategy. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. Finally, we show that the protocol can readily be used to generate homogenous datasets for bulk sequencing of murine bone marrow samples. Taken together, this approach will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

Abstract 4522: An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples

Abstract FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [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 4522.