Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes

Abstract

BackgroundMassively 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.ResultsWe 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.ConclusionWe 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.

Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

“…This process produces 225,623 segregating SNPs, of which 25,757 met our call rate and minor allele frequency criteria for further study (see Methods). Sequence-based SNP calling in P. falciparum is technically challenging because of its extremely AT-rich genome (12,13). In light of this finding, we validated our sequence-based approach against array-based methods by using a previously described SNP array (6) to genotype 24 of the 45 isolates.…”

Diversification in wild populations of the model organism Anolis carolinensis: A genome‐wide phylogeographic investigation

“…(2015), reads passing the high chastity CASAVA filter were assembled as follows. After merging overlapping reads (Rokyta, Lemmon, Margres, & Aronow, 2012), reads were assembled using A. carolinensis references derived from the Vertebrate v1 probe design (Lemmon et al., 2012). Resulting consensus sequences were phased in a Bayesian framework using reads overlapping with polymorphic sites, as described by Pyron et al.…”

Efficient Depletion of Host DNA Contamination in Malaria Clinical Sequencing

“…To obtain an average fragment size of ϳ350 bp, the settings used were 10% duty cycle, intensity 4, and 200 cycles per burst. Illumina paired-end sequencing libraries were constructed using an NEBNext DNA sample preparation kit (New England BioLabs) following the standard Illumina sample preparation protocol (13). All PCR library amplifications, after adapter ligation, were performed with an MJ Research PTC-225 thermocycler.…”

“…Attempts to sequence these samples without adequate removal of the host DNA negate some of the cost benefit realized by the current advances in sequencing technology (11). Whereas problems associated with the small amounts of starting material have been addressed extensively through the development of alternative library preparation methods as well as the discovery of novel amplification technologies (12)(13)(14), host contamination has remained a big challenge, especially for pathogens that are very difficult to culture in vitro.…”