RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

Abstract

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations.

Genetic Drivers of Pancreatic Islet Function

“…At this LOD threshold, 68% of the eQTL were local, suggesting that genetic variation proximal to the gene locus plays a key role in regulating transcript abundance among DO mice. LOD scores for local-eQTL tended to be higher than for distal-eQTL, consistent with what is normally observed in eQTL studies, and thus the proportion of local-eQTL increases with higher LOD thresholds (WEST et al 2007;MUNGER et al 2014). …”

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63

“…Although diverse programs were developed to quantify gene expression levels using mRNA sequencing, alignment of sequencing data of a hybrid is still challenging, as the hybrid contains the genomes of its two parents (62). A previous study showed that constructing individualized diploid genomes and transcriptomes by merging the parental genomes and transcriptomes for the hybrid improved the accuracy of quantification of gene expression levels compared with alignments to a single reference genome (61).…”

Buffering of Genetic Regulatory Networks inDrosophila melanogaster

“…Genotype-specific references have been used to reduce map bias (Degner et al 2009;Rozowsky et al 2011;Graze et al 2012;Yuan and Qin 2012;Stevenson et al 2013;LeonNovelo et al 2014;Munger et al 2014) by incorporating a filtered set of SNPs and indels that were identified in each parental strain. The GATK UnifiedGenotyper tool (ver.…”

“…Masking SNPs and extensive filtering may ameliorate some sources of potential bias (Stevenson et al 2013;van de Geijn et al 2014), but bias may come from multiple sources and not all of these can be effectively filtered (Degner et al 2009;Leon-Novelo et al 2014;van de Geijn et al 2014). There is mounting evidence that using personal genomes is more effective at reducing bias, particularly from structural variants (Degner et al 2009;Rozowsky et al 2011;Graze et al 2012;Yuan and Qin 2012;Munger et al 2014;Zou et al 2014). Further, previously unidentified structural variation can be identified and the bias corrected using appropriate DNA controls (reviewed by Wittkopp et al 2004;Pastinen 2010;Graze et al 2012) and it is impossible to detect and filter such bias without DNA controls.…”