Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length

Barbara Iadarola,Elisabetta Fortunati,Marta Paterno,Renzo Guerrini,Massimo Delledonne,Luciano Xumerle,Cristina Beltrami,Luca Marcolungo,Marzia Rossato,Davide Mei,Denise Lavezzari,Annalisa Vetro,Elena Parrini

doi:10.1038/s41598-020-66331-z

Barbara Iadarola, Elisabetta Fortunati + Show 11 more

Open Access

https://doi.org/10.1038/s41598-020-66331-z

Copy DOI

Abstract

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglected. This work aims to improve the alignment of multiple-mapping reads through the extension of the standard DNA fragment size. As Illumina can sequence fragments up to 550 bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability. This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. Results confirmed that this approach enlightened many regions previously not explored.

Highlights

The exome contains many obscure regions difficult to explore with current short-read sequencing methods
We evaluated the frequency of duplicates and the number of sequenced bases near and off the target obtained using different DNA fragment lengths
Depth of coverage is the parameter used most often to evaluate the performance of Whole-exome sequencing (WES) enrichment technologies, which are applied during the Next-generation sequencing (NGS) of selected target regions in the genome[6]

Summary

Introduction

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. As Illumina can sequence fragments up to 550 bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. In order to rescue variants in well-covered regions with a low mapping quality, base calling (genotypability) calculated by combining the confidence of read alignment with the depth of coverage should be considered as a more informative parameter for the assessment of WES performance[17]. More amplification is necessary, producing more PCR duplicates that must be removed during downstream data analysis, limiting the depth of coverage at target regions[24]

Objectives

Methods

Results

Conclusion