Abstract
Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects.
Highlights
Next-generation sequencing (NGS) has become the leading method for analyzing the architecture of human genomes
Our assay for post-capture exome enrichment validation is composed of 20 Multiplex Ligation-dependent Probe Amplification (MLPA) probes, including 10 probes located in targeted genomic regions, 9 probes located in nontargeted genomic regions, and one probe located in flank of the targeted regions (49 bp from exon 1 of the BARD1 gene)
To allow the direct comparison of the enrichment efficiency of targeted and non-targeted regions situated in close proximity to each other, in two cases, probes of different types were located in the same gene, i.e., BARD1 and ARID1A (Figure 2A)
Summary
Next-generation sequencing (NGS) has become the leading method for analyzing the architecture of human genomes. The cost of whole genome sequencing (WGS) has decreased significantly in recent years, it still substantially hampers the use of WGS for large-scale studies involving abundant DNA sample sets [1]. The use of a targeted enrichment sequencing strategy focused on well-characterized coding sequences, i.e., whole exome sequencing (WES), yields informative results that are easier to interpret [4,5,6]. Targeted/exome sequencing may be favorable especially for applications that require high-coverage of the analyzed regions for identification of low–frequency sequence variants. Such applications include: identification of somatic mutations in cancer genome, identification of mosaic mutations in disease-related genes, identification of mitochondrial DNA heteroplasmy, or identification of sequence variants in mixed DNA samples (e.g., in forensic genetics). Meienberg and colleagues have revealed that currently available exome-enrichment platforms cannot efficiently capture all known coding exons and emphasized the need of constant evaluation of the updated platform versions [11]
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