Dataset Information

Development of Bioinformatics Pipeline for Analyzing Clinical Pediatric NGS Data.

ABSTRACT: Using an Illumina exome sequencing dataset generated from pediatric Acute Myeloid Leukemia patients (AML; type FLT3/ITD+) a comprehensive bioinformatics pipeline was developed to aid in a better clinical understanding of the genetic data associated with the clinical phenotype. The pipeline starts with raw next generation sequencing reads and using both publicly available resources and custom scripts, analyzes the genomic data for variants associated with pediatric AML. By incorporating functional information such as Gene Ontology annotation and protein-protein interactions, the methodology prioritizes genomic variants and returns disease specific results and knowledge maps. Furthermore, it compares the somatic mutations at diagnosis with the somatic mutations at relapse and outputs variants and functional annotations that are specific for the relapse state.

SUBMITTER: Crowgey EL

PROVIDER: S-EPMC4525226 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of Bioinformatics Pipeline for Analyzing Clinical Pediatric NGS Data.

Crowgey Erin L EL Kolb Anders A Wu Cathy H CH

AMIA Joint Summits on Translational Science proceedings. AMIA Joint Summits on Translational Science 20150323

Using an Illumina exome sequencing dataset generated from pediatric Acute Myeloid Leukemia patients (AML; type FLT3/ITD+) a comprehensive bioinformatics pipeline was developed to aid in a better clinical understanding of the genetic data associated with the clinical phenotype. The pipeline starts with raw next generation sequencing reads and using both publicly available resources and custom scripts, analyzes the genomic data for variants associated with pediatric AML. By incorporating functiona ...[more]

PMID: 26306272

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Project description:Diazotrophic microorganisms introduce biologically available nitrogen (N) to the global N cycle through the activity of the nitrogenase enzyme. The genetically conserved dinitrogenase reductase (nifH) gene is phylogenetically distributed across four clusters (I-IV) and is widely used as a marker gene for N2 fixation, permitting investigators to study the genetic diversity of diazotrophs in nature and target potential participants in N2 fixation. To date there have been limited, standardized pipelines for analyzing the nifH functional gene, which is in stark contrast to the 16S rRNA gene. Here we present a bioinformatics pipeline for processing nifH amplicon datasets - NifMAP ("NifH MiSeq Illumina Amplicon Analysis Pipeline"), which as a novel aspect uses Hidden-Markov Models to filter out homologous genes to nifH. By using this pipeline, we evaluated the broadly inclusive primer pairs (Ueda19F-R6, IGK3-DVV, and F2-R6) that target the nifH gene. To evaluate any systematic biases, the nifH gene was amplified with the aforementioned primer pairs in a diverse collection of environmental samples (soils, rhizosphere and roots samples, biological soil crusts and estuarine samples), in addition to a nifH mock community consisting of six phylogenetically diverse members. We noted that all primer pairs co-amplified nifH homologs to varying degrees; up to 90% of the amplicons were nifH homologs with IGK3-DVV in some samples (rhizosphere and roots from tall oat-grass). In regards to specificity, we observed some degree of bias across the primer pairs. For example, primer pair F2-R6 discriminated against cyanobacteria (amongst others), yet captured many sequences from subclusters IIIE and IIIL-N. These aforementioned subclusters were largely missing by the primer pair IGK3-DVV, which also tended to discriminate against Alphaproteobacteria, but amplified sequences within clusters IIIC (affiliated with Clostridia) and clusters IVB and IVC. Primer pair Ueda19F-R6 exhibited the least bias and successfully captured diazotrophs in cluster I and subclusters IIIE, IIIL, IIIM, and IIIN, but tended to discriminate against Firmicutes and subcluster IIIC. Taken together, our newly established bioinformatics pipeline, NifMAP, along with our systematic evaluations of nifH primer pairs permit more robust, high-throughput investigations of diazotrophs in diverse environments.

Project description:BACKGROUND:Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses. METHODS:Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail. RESULTS:In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%. CONCLUSIONS:We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.

Dataset Information

Development of Bioinformatics Pipeline for Analyzing Clinical Pediatric NGS Data.

Publications

Development of Bioinformatics Pipeline for Analyzing Clinical Pediatric NGS Data.

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