Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes
By Samuel O. Oyola, Thomas D. Otto, Yong Gu, G. Maslen, M. Manske, Susana G. Campino, D. Turner, B. MacInnis, Dominic Kwiatkowski, H. Swerdlow and 1 more. BMC Genomics, 2012
481 citations recorded by the source corpus.
Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.
Subfields: Epidemiology, Infectious Diseases, Public Health, Environmental and Occupational Health
View Paper: https://www.semanticscholar.org/paper/df5302927aeb1bb1e7237901db0deb4862601db7
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Citations in the corpus
- Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies (2020, methodology): “Although we currently recommend using Illumina data for the k-mer-based validation, it may require special library preparation to minimize sequencing biases [40 – 42].”
- Recombinase Polymerase Amplification for Diagnostic Applications (2016, background): “RPA speed could be potentially exploited for NGS (next-generation sequencing) applications for presequencing amplification required for generating libraries (64).”
- Characterizing and measuring bias in sequence data (2013, methodology): “PCR amplification during library construction is a known source of undercoverage of GC-extreme regions [20,21] and similar biases may also be introduced during bridge PCR for cluster amplification on the Illumina flowcell [23].”