Bioinformatics Pipeline for Next Generation Sequencing Analysis in Association Studies of Idiopathic Pulmonary Fibrosis

Abstract

A complete bioinformatics pipeline for Next Generation Sequencing (NGS) analysis has been developed and applied to study the association of called variants with susceptibility in Idiopathic Pulmonary Fibrosis (IPF). This bioinformatics pipeline integrates the Genome Analysis Toolkit (GATK) with state-of-the-art bioinformatics tools such as quality control reporters, aligners, alternative callers (i.e. Platypus), annotators, and auxiliary tools. The pipeline executes a sequence of SBash and Bash shell scripts by queuing the programmed jobs to a SLURM queue at a cluster server provided by La Laguna University (ULL). It is also executable with a local Linux machine. We tested the pipeline by calling single nucleotide polymorphisms (SNPs) in targeted NGS data from 192 individuals with IPF, where 16,253 variant sites were identified. The call concordance between the two utilized callers (GATK and Platypus) was estimated at 77.8% when we compared matching overlapping sites. With this data, an association study following an unmatched case-control design was performed using unrelated European individuals (n=501) from The 1000 Genomes Project as controls. Logistic regression models were applied to the phenotype trait using genotypes from the 10,245 SNPs with call rates >95%, adjusting with five principal components to account for population stratification. Despite the reduced sample size, we identified 38 variants reaching genome-wide significance (p<5x10-8), including one previously identified in the promoter region of MUC5B gene (rs35705950), and several other novel susceptibility variants.