Complex Indel Research Articles

Abstract 340: Validation of TSO500 NGS panel for comprehensive genomic profiling from FFPE tumor samples

Abstract Comprehensive genomic profiling (CGP) of tumor sample using large targeted NGS panels (e.g. MSK-IMPACT) has been shown to allow the detection of biomarkers from hundreds of cancer relevant genes involved in targeted and immunotherapy. Illumina TruSight Oncology 500 (TSO500), a commercial CGP assay, allows concurrent analysis of DNA and RNA to simultaneously detect multiple types of variants across 523 genes. The assay is intended to detect single nucleotide variants (SNV), insertions and deletions (indel), and multinucleotide variants (MNV), copy number gain (CNV), tumor mutational burden (TMB) and microsatellite instability (MSI) in DNA, and fusions and splicing variants (exon skipping) in RNA. Here we aimed to validate TSO500 assay for its analytical performance on FFPE tumor samples. The validation samples include commercial controls, cell lines, clinical FFPE from NSCLC, CRC, Uterus and GIST (tumor content ≥20%). Library preparation was performed from minimum of 40 ng DNA and 40ng RNA. The data analysis is performed using TruSight Oncology 500 Local App software (LRM v2.2). A custom script (v1.0) was used to add SNP annotation for small variants and combine data output into cumulative format. For SNV and indel detection QC, 61 of 64 DNA libraries (95%) passed the QC for library size (median insert size ≥70bp) and coverage (median exon coverage ≥150). The percentage of DNA libraries passing QC for CNV and MSI are 95% and 88%, respectively. All 64 RNA libraries passed QC. The analytical performance of TSO500 was summarized in the Table below. In summary, the results showed that all the acceptance criteria were met and the TSO500 assay can be utilized for genomic profiling of FFPE tumor samples. The study also identified a few limitations of the Illumina LRM software e.g. potential false positive SNV within a indel background, potential false negative of complex indel, and the challenge of long indel detection. Summary of analytical performance of TSO500 Sensitivity and specificity Assay LoD Accuracy by concordance Inter-run reproducibility SNV/Small indel Sensitivity for SNV and indel detection are 99.24% and 99.36%. The LoD for SNV is 3-5%, for DEL is 5-10% and for INS is 3-5%. PPA for SNV detections is 96.64% (115/119; 95% CI: 91.68%, 98.69%) and for small indel is 92.86% (13/14; 95% CI: 68.53%, 98.73%) between TSO500 and comparator assays. The positive call rate across all SNV and small indel variants is 98.7% (42842/43419). Specificity for SNV and small indel detection are 99.9998% and 100.00%, respectively. MSI Sensitivity and Specificity for MSI detection are both 100%. NA MSI status for all samples were detected with 100% of concordance by three assays; MSI concordance for MSI-H is 100% (95% CI: 60.97%, 100.0%) and for MSS is 100% (87.13%, 100.0%). PPA is 100% (95% CI: 51.01%, 100%) and NPA is 100% (95% CI: 34.24%, 100%). TMB NA NA TSO500 reported TMB is well correlated and comparable to exome determined TMB. The CV values for samples are acceptable. CNV NA NA All positive CNVs detected by the comparator assay were detected by the TSO500 assay with similar fold changes; The overall reproducible call rate is 91.5% (43/47). PPA between TSO500 and OCAV3 assays is 100% (95% CI: 51.01%, 100%). Fusion Sensitivity and specificity for fusion detection is 91.94% and 100%, respectively. NA All TSO500 assay detected fusions were confirmed by other assays;PPA is 100% (95% CI: 60.97%, 100%) and NPA is 100% (95% CI: 75.75%, 100%) The reproducibility of fusion call is 92.59% (25/27; 95% CI: 76.63%, 97.94%). Splicing Sensitivity and Specificity for splicing variant detection are 91.67% and 100.00%, respectively. NA The splicing variant detections were confirmed by OCAV3, OFA and Archer assays; The overall reproducibility of positive call is 100% with 95% CI (43.85%, 100%) and negative call reproducibility is 100% with 95% CI (87.13%, 100%). PPA is 100% (95%% CI: 20.65%, 100%) and NPA is 100% (95% CI: 83.18%, 100%) Citation Format: Jin Li, Zhenyu Yan, Weihua Liu, Lin Shi, Cynthia Spittle, Chad Galderisi. Validation of TSO500 NGS panel for comprehensive genomic profiling from FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 340.

A biallelic frameshift indel in PPP1R35 as a cause of primary microcephaly.

Protein phosphatase 1 regulatory subunit 35 (PPP1R35) encodes a centrosomal protein required for recruiting microtubule-binding elongation machinery. Several proteins in this centriole biogenesis pathway correspond to established primary microcephaly (MCPH) genes, and multiple model organism studies hypothesize PPP1R35 as a candidate MCPH gene. Here, using exome sequencing (ES) and family-based rare variant analyses, we report a homozygous, frameshifting indel deleting the canonical stop codon in the last exon of PPP1R35 [Chr7: c.753_*3delGGAAGCGTAGACCinsCG (p.Trp251Cysfs*22)]; the variant allele maps in a 3.7Mb block of absence of heterozygosity (AOH) in a proband with severe MCPH (-4.3 SD at birth, -6.1 SD by 42 months), pachygyria, and global developmental delay from a consanguineous Turkish kindred. Droplet digital PCR (ddPCR) confirmed mutant mRNA expression in fibroblasts. In silico prediction of the translation of mutant PPP1R35 is expected to be elongated by 18 amino acids before encountering a downstream stop codon. This complex indel allele is absent in public databases (ClinVar, gnomAD, ARIC, 1000 genomes) and our in-house database of 14,000+ exomes including 1800+ Turkish exomes supporting predicted pathogenicity. Comprehensive literature searches for PPP1R35 variants yielded two probands affected with severe microcephaly (-15 SD and -12 SD) with the same homozygous indel from a single, consanguineous, Iranian family from a cohort of 404 predominantly Iranian families. The lack of heterozygous cases in two large cohorts representative of the genetic background of these two families decreased our suspicion of a founder allele and supports the contention of a recurrent mutation. We propose two potential secondary structure mutagenesis models for the origin of this variant allele mediated by hairpin formation between complementary GC rich segments flanking the stop codon via secondary structure mutagenesis.

DelInsCaller: An Efficient Algorithm for Identifying Delins and Estimating Haplotypes from Long Reads with High Level of Sequencing Errors.

Delins, as known as complex indel, is a combined genomic structural variation formed by deleting and inserting DNA fragments at a common genomic location. Recent studies emphasized the importance of delins in cancer diagnosis and treatment. Although the long reads from PacBio CLR sequencing significantly facilitate delins calling, the existing approaches still encounter computational challenges from the high level of sequencing errors, and often introduce errors in genotyping and phasing delins. In this paper, we propose an efficient algorithmic pipeline, named delInsCaller, to identify delins on haplotype resolution from the PacBio CLR sequencing data. delInsCaller design a fault-tolerant method by calculating a variation density score, which helps to locate the candidate mutational regions under a high-level of sequencing errors. It adopts a base association-based contig splicing method, which facilitates contig splicing in the presence of false-positive interference. We conducted a series of experiments on simulated datasets, and the results showed that delInsCaller outperformed several state-of-the-art approaches, e.g., SVseq3, across a wide range of parameter settings, such as read depth, sequencing error rates, etc. delInsCaller often obtained higher f-measures than other approaches; specifically, it was able to maintain advantages at ~15% sequencing errors. delInsCaller was able to significantly improve the N50 values with almost no loss of haplotype accuracy compared with the existing approach as well.

IndelPost: harmonizing ambiguities in simple and complex indel alignments.

Small insertions and deletions (indels) in nucleotide sequence may be represented differently between mapping algorithms and variant callers, or in the flanking sequence context. Representational ambiguity is especially profound for complex indels, complicating comparisons between multiple mappings and call sets. Complex indels may additionally suffer from incomplete allele representation, potentially leading to critical misannotation of variant effect. We present indelPost, a Python library that harmonizes these ambiguities for simple and complex indels via realignment and read-based phasing. We demonstrate that indelPost enables accurate analysis of ambiguous data and can derive the correct complex indel alleles from the simple indel predictions provided by standard small variant detectors, with improved performance over a specialized tool for complex indel analysis. indelPost is freely available at: https://github.com/stjude/indelPost. Supplementary data are available at Bioinformatics online.

Two novel intragenic variants in the FMR1 gene in patients with suspect clinical diagnosis of Fragile X syndrome and no CGG repeat expansion

The major and most well-studied genetic cause of Fragile-X syndrome (FXS) is expansion of a CGG repeat in the 5′-UTR of the FMR1 gene. Routine testing for this expansion is performed globally. Overall, there is a paucity of intragenic variants explaining FXS, a fact which is being addressed by a more systematic application of whole exome (WES) and whole genome (WGS) sequencing, even in the diagnostic setting. Here we report two families comprising probands with a clinical suspicion of FXS and no CGG repeat expansions. Using WES/WGS we identified deleterious variants within the coding region of FMR1 in both families. In a family from Finland we identified a complex indel c.1021-1028delinsTATTGG in exon 11 of FMR1 which gives rise to a frameshift and a premature termination codon (PTC), p.Asn341Tyrfs*7. Follow-up mRNA and protein studies on a cell line from the proband revealed that although the mRNA levels of FMR1 were not altered, Fragile X Mental Retardation 1 Protein (FMRP) was undetectable. Additionally, we identified a variant, c.881-1G > T, affecting the canonical acceptor splice site of exon 10 of FMR1 in an Australian family. Our findings reinforce the importance of intragenic FMR1 variant testing, particularly in cases with clinical features of FXS and no CGG repeat expansions identified.

Abstract 5294: CIMerge: A machine learning approach for merging and genotyping complex indel calls from NGS data

Abstract Complex insertion and deletion (complex indel) is a rare category of genomic structural variations, which is formed by inserting one or multiple DNA fragments into the genomic location where a deletion occurs. A recent study conducts a systematic analysis from over 8,000 pan-cancer cases, which reports hundreds of complex indels in cancer-associated genes, some of which are considered potentially druggable. Several approaches are proposed to detect complex indels from the new-generation sequencing data (both 2nd and 3rd generation). However, as different data-mining algorithms vary the preferences on capturing data patterns, different approaches may report the conflicted complex indel calls. Here, we propose a machine learning approach to correct the conflicted calls from different approaches and further estimate the genotype of each call. The proposed approach, implemented as CIMerge, adopts a relevance vector machine framework. For each candidate call, CIMerge first extracts a set of features on the candidate region, which includes the read depth, variant allelic frequency, number of the splitting/unmapped reads, number of the discordant paired-end reads, aligned contig, etc. CIMerge also considers another set of features on data-mining algorithm(s) that reported the candidate call, which includes the parameter settings, etc. Both sets of features are trained by the relevance vector machine framework, which outputs the probability of each candidate of the conflicted call. As a byproduct, it outputs the genotype of the candidate call with highest likelihood. We tested CIMerge on multiple datasets generated by different simulation configurations and compared it to several state-of-the-art approaches. The experiment results demonstrate that CIMerge outperforms the existing approaches. The average success rate of recognition is approaching 90%, while Pindel and Gindel are reported as 62.53% and 65.202%, respectively. The software package CIMerge is freely available for academic uses at https://github.com/xjtu712-lab/CIMerge. Citation Format: Tian Zheng, Yang Li, Yu Geng, Zhongmeng Zhao, Xuanping Zhang, Jiayin Wang. CIMerge: A machine learning approach for merging and genotyping complex indel calls from NGS data [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 5294.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Zebrafish Model of Diamond-Blackfan Anemia Results in Bone Marrow Failure and Demonstrates Defective Translation in Erythroid Cells By Ribosome Footprinting

Diamond-Blackfan anemia (DBA) is a congenital bonemarrow failure syndrome that manifests as a profoundmacrocytic anemia, which classically presents within the first year of life. Heterozygous mutations or allelic loss of one of 12 ribosomal proteins (RP) leads to apoptosis of erythroid precursors and have been identified in over 50% of DBA patients, most commonly RPS19 that accounts for 25% of all cases.To study the role of Rps19 in erythroid development, we employed genome-editing tools to generate stable knockout line of Rps19 in zebrafish using TALENs targeting exon 1 of Rps19. We generated a stable mutant line and these mutants have a 5 bp complex indel in Exon 1 resulting in N13fs and truncated protein. Rps19-/- embryos show profound developmental anomalies including a marked anemia and are embryonic lethal by 5dpf.Rps19+/- heterozygotes were indistinguishable from their WT siblings during early development and had no erythroid defect detectable by whole mount in situ hybridization (WISH) or histochemical staining. To assess the effects of Rps19 on embryonic erythropoiesis more detail, we used Rps19 mutants carrying the Tg(gata1:dsRed) and Tg(globin:eGFP) transgenes. Rps19+/- fish were crossed and then exposed to cold stress from 12 hours post fertilization(hpf) continuously (22 degrees) and then analyzed by flow cytometry at the developmental age of 96hpf. GFP, dsRed and double GFP/dsRed expressing erythroid cells showed a reduction in cell number in Rps19+/- compared to Rps19+/+ siblings. Therefore, when stress is induced, the loss of one copy of rps19 is sufficient to induce anemia.To further study the effects of Rps19 on erythroid development, we investigated the phenotype of adult fish. Adult Rps19+/- fish at 6 months of age were significantly smaller than their siblings, weighed less and showed reduced hemoglobin levels in their peripheral blood. We then further characterized the adult rps19-deficient zebrafish phenotype by flow cytometry of whole kidney marrow using Tg(globin:eGFP);Rps19+/- animals. Rps19+/ - adults demonstrated pancytopenia, with a marked decrease in the erythroid lineage, similar to the phenotype observed in DBA patients.Recent data have suggested that tissue specific functions of ribosomes are common and we hypothesized that such an effect may explain the tropism of Rps19+/- phenotypes seen in DBA to erythroid and hematopoietic tissue. To test this hypothesis we assessed the effect of translation in Rps19+/- erythroid cells compared to Rps19+/+ using genome-wide analysis of translation by ribosome footprinting (RF) on sorted GFP+ cells from whole kidney marrow using Tg(globin:eGFP);Rps19 mutantzebrafish. In parallel we assessed RNAseq from the same samples.RNAseq showed differential expression of genes involved in mitochondrial cell death pathways including upregulation of caspase 9 and downregulation of p53 . Stabilization of p53 and consequent p53-dependent apoptosis is a well recognized mechanism of the anemia in DBA, however our data suggest that such stabilization results in transcriptional downregulation of p53 as a compensatory mechanism to maintain a steady state level of erythropoiesis. RF data by contrast showed that the principle genes with aberrant translation in Rps19+/- erythroid cells are other ribosomal protein genes, predominantly (although not exclusively) of the large subunit. In addition we observed that one of the most markedly upregulated footprints was on the branched chain keto acid dehydrogenase E1 subunit beta gene (encoded by bckdhb). This enzyme is part of a multimolecular complex that resides on the inner membrane of the mitochondria and is responsible for catabolism of branched chain amino acids L-Leucine and Valine, highlighting a potential underlying mechanism by which erythroid cells may be functionally leucine deficient, and thus respond to exogenous L-leucine as has previously been shown in zebrafish, murine and human cell models.In summary, we have generated a stable Rps19 mutant line and characterized the phenotype of heterozygous Rps19+/- mutants, which recapitulate features of DBA during developmental and adult hematopoiesis. In addition, we have used ribosome footprinting to study the effect of Rps19 heterozygosity on translation within erythroid cells of different genes, providing new insight for potential drug targets. Our on-going work is further studying the differentially expressed genes. DisclosuresNo relevant conflicts of interest to declare.

Sensitivity of whole exome sequencing in detecting infantile- and late-onset Pompe disease.

Pompe disease is a metabolic myopathy with a wide spectrum of clinical presentation. The gold-standard diagnostic test is acid alpha-glucosidase assay on skin fibroblasts, muscle or blood. Identification of two GAA pathogenic variants in-trans is confirmatory. Optimal effectiveness of enzyme replacement therapy hinges on early diagnosis, which is challenging in late-onset form of the disease due to non-specific presentation. Next-generation sequencing-based panels effectively facilitate diagnosis, but the sensitivity of whole-exome sequencing (WES) in detecting pathogenic GAA variants remains unknown. We analyzed WES data from 93 patients with confirmed Pompe disease and GAA genotypes based on PCR/Sanger sequencing. After ensuring that the common intronic variant c.-32-13T>G is not filtered out, whole-exome sequencing identified both GAA pathogenic variants in 77/93 (83%) patients. However, one variant was missed in 14/93 (15%), and both variants were missed in 2/93 (2%). One complex indel leading to a severe phenotype was incorrectly called a nonsynonymous substitution c.-32-13T>C due to misalignment. These results demonstrate that WES may fail to diagnose Pompe disease. Clinicians need to be aware of limitations of WES, and consider tests specific to Pompe disease when WES does not provide a diagnosis in patients with proximal myopathy, progressive respiratory failure or other subtle symptoms.

Abstract 3592: Improved indel detection in RNA-seq data via assembly based re-alignment reveals expressed Epidermal Growth Factor Receptor indels in Lung Adenocarcinoma

Abstract Insertions and deletions in the transcriptome can potentially have significant impact on function and can be clinically actionable. A number of methods have recently been developed that improve indel detection in DNA by utilizing realignment and/or localized assembly to aid in discovering mutations that are more difficult to detect than single nucleotide polymorphisms. While significant effort has been put into these methods in the context of DNA discovery, RNA-Seq has not received the same attention. For example, the widely used Genome Analysis Toolkit (GATK) recommends removal of splice junctions from all reads prior to proceeding with variant calling methods that are very similar to those used for DNA. We hypothesize that utilizing splice junctions to augment localized assembly across exon-exon boundaries can improve read alignments resulting in improved variant detection in RNA-Seq data. We have developed an update to the Assembly Based ReAligner (ABRA2) that makes use of splice junction information to aid in realignment of reads. We assessed indel detection performance using the BEERs RNA-Seq simulator. Two million simulated reads of length 100nt were generated across 1000 human genes. In order to test more complex indel detection, the simulator was modified to generate an even distribution of 1391 indels of length 1 to 100. The simulated reads were aligned using STAR and subsequently realigned using ABRA2. Freebayes and GATK were run against the non-realigned STAR output resulting in indel detection sensitivity of 15% and 18% with precision of 97% and 88% respectively. Freebayes was then run against the ABRA2 realignments resulting in an approximately 4 fold increase in indel detection sensitivity of 67% while maintaining precision of 97%. In frame deletions in Epidermal Growth Factor Receptor (EGFR) have oncogenic potential, can be indicators for Gefitinib or Erlotinib treatment, and are frequently detected in lung cancer via DNA sequencing. Among 514 TCGA Lung Adenocarcinoma RNA-Seq samples, ABRA2 revealed 73 in frame coding indels in EGFR ranging in length from 3 to 24 bases, including 58 deletions in exon 19 of length 15 bases. This represents a 3 fold increase relative to what was initially reported from DNA by TCGA. Further, in the absence of ABRA2, only 5 in frame coding indels are detected in RNA, the largest of which is a 9 base deletion. We have presented here ABRA2, a new version of the Assembly Based ReAligner that is capable of accurately realigning RNA-Seq reads containing variations that are currently not well handled by widely used aligners and variant callers, thus improving accuracy of variant detection in RNA-Seq. Citation Format: Lisle E. Mose, D. Neil Hayes, Charles M. Perou, Joel S. Parker. Improved indel detection in RNA-seq data via assembly based re-alignment reveals expressed Epidermal Growth Factor Receptor indels in Lung Adenocarcinoma [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 3592. doi:10.1158/1538-7445.AM2017-3592

Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein.

Proteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single—amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

INDELseek: detection of complex insertions and deletions from next-generation sequencing data

BackgroundComplex insertions and deletions (indels) from next-generation sequencing (NGS) data were prone to escape detection by currently available variant callers as shown by large-scale human genomics studies. Somatic and germline complex indels in key disease driver genes could be missed in NGS-based genomics studies.ResultsINDELseek is an open-source complex indel caller designed for NGS data of random fragments and PCR amplicons. The key differentiating factor of INDELseek is that each NGS read alignment was examined as a whole instead of “pileup” of each reference position across multiple alignments. In benchmarking against the reference material NA12878 genome (n = 160 derived from high-confidence variant calls), GATK, SAMtools and INDELseek showed complex indel detection sensitivities of 0%, 0% and 100%, respectively. INDELseek also detected all known germline (BRCA1 and BRCA2) and somatic (CALR and JAK2) complex indels in human clinical samples (n = 8). Further experiments validated all 10 detected KIT complex indels in a discovery cohort of clinical samples. In silico semi-simulation showed sensitivities of 93.7–96.2% based on 8671 unique complex indels in >5000 genes from dbSNP and COSMIC. We also demonstrated the importance of complex indel detection in accurately annotating BRCA1, BRCA2 and TP53 mutations with gained or rescued protein-truncating effects.ConclusionsINDELseek is an accurate and versatile tool for complex indel detection in NGS data. It complements other variant callers in NGS-based genomics studies targeting a wide spectrum of genetic variations.

Systematic discovery of complex insertions and deletions in human cancers.

Complex indels are formed by simultaneously deleting and inserting DNA fragments of different sizes at a common genomic location. Here, we present a systematic analysis of somatic complex indels in the coding sequences of over 8,000 cancer cases using Pindel-C. We discovered 285 complex indels in cancer genes (e.g., PIK3R1, TP53, ARID1A, GATA3, and KMT2D) in approximately 3.5% of cases analyzed; nearly all instances of complex indels were overlooked (81.1%) or mis-annotated (17.6%) in 2,199 samples previously reported. In-frame complex indels are enriched in PIK3R1 and EGFR while frameshifts are prevalent in VHL, GATA3, TP53, ARID1A, PTEN, and ATRX. Further, complex indels display strong tissue specificity (e.g., VHL from kidney cancer and GATA3 from breast cancer). Finally, structural analyses support findings of previously missed, but potentially druggable mutations in EGFR, MET, and KIT oncogenes. This study indicates the critical importance of improving complex indel discovery and interpretation in medical research.

Insertion - Deletion as Informative Characters in DNA Barcoding

DNA barcoding involve rapidly sequencing the standardized region from the genome for species identification and this homologous sequence information plays an important role to resolve the phylogenetic issues. However in case of complex groups, incorporation of insertion and deletion (Indel) informative sites in DNA barcode sequences are becoming more important due to low substitution rate. Indels are largely ignored in phylogenetics analysis and removed from sequence alignment assigning them a missing data. We review the current trends on mining the indels analysis, focusing especially on the topics of rapidly evolving indel containing loci and methods of indel treatment for phylogenetic relationship. Among the indel coding methods, Simple indel coding is easy to implement in indels contain sequences. But, this method does not utilized the all the available information and Complex Indel Coding rules are very difficult to translate into a clearly formulate algorithm for determining the values. But this coding method suffers from internal inconsistence when a long indel has a number of shorter subset indel and triangular in equality exists. However, SIDIER software package infer evolutionary relationships based on both the indels and substitutions. SIDIER is promising software for intra and inter specific calculation in DNA barcode studies as well as to infer phylogenetic relationships.

Highly Sensitive Diagnosis of 43 Monogenic Forms of Diabetes or Obesity Through One-Step PCR-Based Enrichment in Combination With Next-Generation Sequencing

Accurate etiological diagnosis of monogenic forms of diabetes and obesity is useful as it can lead to marked improvements in patient care and genetic counseling. Currently, molecular diagnosis based on Sanger sequencing is restricted to only a few genes, as this technology is expensive, time-consuming, and labor-intensive. High-throughput next-generation sequencing (NGS) provides an opportunity to develop innovative cost-efficient methods for sensitive diabetes and obesity multigene screening. We assessed a new method based on PCR enrichment in microdroplets (RainDance Technologies) and NGS using the Illumina HiSeq2000 for the molecular diagnosis of 43 forms of monogenic diabetes or obesity. Forty patients carrying a known causal mutation for those subtypes according to diagnostic laboratories were blindly reanalyzed. Except for one variant, we reidentified all causal mutations in each patient associated with an almost-perfect sequencing of the targets (mean of 98.6%). We failed to call one highly complex indel, although we identified a dramatic drop of coverage at this locus. In three patients, we detected other mutations with a putatively deleterious effect in addition to those reported by the genetic diagnostic laboratories. Our NGS approach provides an efficient means of highly sensitive screening for mutations in genes associated with monogenic forms of diabetes and obesity. As cost and time to deliver results have been key barriers to uncovering a molecular cause in the many undiagnosed cases likely to exist, the present methodology should be considered in patients displaying features of monogenic diabetes or obesity.

A frameshift mutation in the melanophilin gene causes the dilute coat colour in rabbit (Oryctolagus cuniculus) breeds

In rabbit, the dilute locus is determined by a recessive mutated allele (d) that causes the dilution of both eumelanic and pheomelanic pigmentations. In mice, similar phenotypes are determined by mutations in the myosin VA, Rab27a and melanophilin (MLPH) genes. In this study, we investigated the rabbit MLPH gene and showed that a mutation in this gene appears responsible for the dilute coat colour in this species. Checkered Giant F1 families segregating for black and grey (diluted or blue) coat colour were first genotyped for a complex indel in intron 1 of the MLPH gene that was completely associated with the coat colour phenotype (θ = 0.00; LOD = 4.82). Then, we sequenced 6357 bp of the MLPH gene in 18 rabbits of different coat colours, including blue animals. A total of 165 polymorphisms were identified: 137 were in non-coding regions and 28 were in coding exons. One of them was a frameshift deletion in exon 5. Genotyping the half-sib families confirmed the complete cosegregation of this mutation with the blue coat colour. The mutation was analysed in 198 rabbits of 23 breeds. All Blue Vienna and all other blue/grey/ash rabbits in other breeds (Californian, Castor Rex, Checkered Giant, English Spot, Fairy Marburg and Fairy Pearly) were homozygous for this deletion. The identification of MLPH as the responsible gene for the dilute locus in rabbit provides a natural animal model for human Griscelli syndrome type 3 and a new mutant to study the role of this gene on pigmentation.

Characterization of a recurrent in-frame UMOD indel mutation causing late-onset autosomal dominant end-stage renal failure.

In a single-center renal clinic, we have established routine mutation testing to diagnose UMOD-associated kidney disease (UAKD), an autosomal dominant disorder typically characterized by gout, hyperuricemia, and renal failure in the third to sixth decades. Four probands and their multigeneration kindreds were assessed by clinical, historical, and biochemical means. Diagnostic UMOD sequencing was performed, and mutant uromodulin was characterized in vitro. All available affected members of the four kindreds harbored the same complex indel change in UMOD, which was associated with almost complete absence of gout and a later onset of CKD; the youngest age at ESRD or death was 38 years (range, 38 to 68 years) compared with 3 to 70 years in other reports. Three mutation carriers (all ≤35 years) are currently asymptomatic. The indel sequence (c.278_289del TCTGCCCCGAAGinsCCGCCTCCT; p.V93_G97del/ins AASC) results in the replacement of five amino acids, including one cysteine, by four novel residues, also including a cysteine. Uromodulin staining of the only available patient biopsy suggested disorganized intracellular trafficking with cellular accumulation. Functional characterization of the mutant isoform revealed retarded intracellular trafficking associated with endoplasmic reticulum (ER) retention and reduced secretion into cell culture media, but to a lesser extent than we observed with the previously reported C150S mutation. The indel mutation is associated with a relatively mild clinical UAKD phenotype, consistent with our in vitro analysis. UAKD should be routinely considered as a causative gene for ESRD of unknown cause, especially where there is an associated family history or where biopsy reveals interstitial fibrosis.

The relative performance of indel-coding methods in simulations

We used simulations to compare the performance of 10 approaches that have been used for treating unambiguously aligned gaps in phylogenetic analyses. We examined how these approaches perform under the ideal conditions of correct alignments, as well as how robust they are to errors caused by use of inferred alignments. Our results indicate that 5th-state coding dramatically outperformed all other coding methods, which in turn all outperformed treating gaps as missing data or excluding gapped positions. Simple indel coding (SIC) and modified complex indel coding (MCIC) performed about the same, and generally outperformed the other indel-coding methods. The high performance of 5th-state coding was largely found to be a weighting artifact. We suggest that MCIC-coded gap characters be scored for all unambiguously aligned gaps in parsimony-based molecular phylogenetic analyses. When the number of terminals sampled precludes the use of MCIC, SIC may be used as an effective substitute.

Complex insertion/deletion polymorphism in NOD1 (CARD4) is not associated with inflammatory bowel disease susceptibility in East Anglia panel

Genetic association between inflammatory bowel disease (IBD) and NOD1 (CARD4) has recently been reported. This gene has structural similarity to NOD2 (CARD15), a confirmed susceptibility gene for Crohn"s disease (CD). The NOD1 association was strongest at novel complex indel ND1 + 32656. Our aim was to ascertain the contribution of ND1 + 32656 variants to IBD in a large independent United Kingdom dataset and to identify any subphenotype association within CD and ulcerative colitis (UC). The presence of the ND1 + 32656 variant in our panel was confirmed by direct resequencing in 96 cases. One thousand three hundred seventy unrelated white IBD subjects (671UC, 645 CD, 54 indeterminate) and 760 regionally matched controls were then genotyped for the ND1 + 32656 variant. Data were analyzed by logistic regression methods within STATA software. There was no association between ND1 + 32656 and IBD in our panel. There was no heterogeneity between UC and CD, nor within the CD subgroup when conditioned by subphenotype or the presence of NOD2 variants. There was no overall evidence of association between IBD and the reported NOD1 susceptibility variant ND1 + 32656 in our panel. The discrepancy with the earlier report may reflect a smaller effect size than previously predicted, a false-positive result in the index study, or population heterogeneity.

Incorporating information from length-mutational events into phylogenetic analysis

With the growing number of phylogenetic studies that use length variable DNA sequences, incorporating information from length-mutational events into phylogenetic analysis is becoming increasingly important. A new method, modified complex indel coding is described that aims at maximizing the phylogenetic information retained from unambiguously aligned sequence regions or regions where the principal relative position of gaps to one another can be safely established. An algorithm is described that allows application of the method to all theoretically possible gap-nucleotide patterns. A platform-independent computer program is introduced that automates the new method as well as several previously published coding schemes. Differences to previously published indel coding approaches as well as to the integration of ambiguously aligned regions into phylogenetic analysis are discussed.