Open AccessMethod for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology
Author(s) -
Olivier Harismendy,
Kelly A. Frazer
Publication year - 2009
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000113082
Subject(s) - amplicon , amplicon sequencing , biology , insert (composites) , dna sequencing , genetics , sequence (biology) , computational biology , illumina dye sequencing , deep sequencing , polymerase chain reaction , genome , dna , gene , mechanical engineering , 16s ribosomal rna , engineering
One approach for high-throughput population-based sequencing of targeted intervals in the human genome is to amplify the regions using long-range PCR (LR-PCR) followed by sequencing with next-generation sequencing (NGS) technologies. Utilizing this method, we have observed that the 50 bp located at the amplicon ends account for more than 50% of the sequenced bases and that the sequence coverage depth of base pairs within an amplicon is highly variable. Here we propose an explanation for the overrepresentation of the amplicon ends and show that the use of 5′-blocked primers for the LR-PCR reaction reduces their overrepresentation. Furthermore, we demonstrate that using a 600-bp library insert size rather than the standard 200-bp insert size results in more uniform sequence coverage depth. The capability to increase sequence coverage uniformity greatly improves the effective throughput of NGS platforms.