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Approaching Single-Cell Sequencing by Understanding NGS Library Complexity and Bias

Demands are growing on genomics to deliver higher quality sequencing data from samples with less input quantity. As the number of genomic equivalents decreases at lower input amounts, library bias and library complexity increasingly affect data quality. Less biased, more complex libraries result in more even and complete coverage, resulting in better sequencing efficiency and reduced costs. This application note describes the sequence coverage performance and preservation of molecular complexity of next generation sequencing (NGS) libraries generated from human and microbial genomic DNA using the Accel-NGS™ 2S DNA Library Kit for whole-genome sequencing (WGS) on the Illumina® platform. Comparisons to the leading commercially available methods are also presented. From the lowest input DNA quantity supported for PCR-free libraries (100 ng for Accel-NGS 2S vs. 1 μg for the leading kit), this new technology demonstrated more than 50% higher library complexity. Coverage of extreme GC-rich regions was characterized using the 1,000 bad promoters (~79% GC) defined by the Broad Institute. The subsequent sequencing results demonstrated superior coverage performance of these high GC-content promoters, where 88% were covered better with Accel-NGS 2S than the market leading kit. Similarly, sequencing analysis of libraries prepared from AT-rich and GC-rich bacterial genomes showed excellent coverage distribution.

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