Identification of Optimum Sequencing Depth Especially for De Novo Genome Assembly of Small Genomes Using Next Generation Sequencing Data

Abstract

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6–40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Genome analysis of Campylobacter concisus strains from patients with inflammatory bowel disease and gastroenteritis provides new insights into pathogenicity

“…The reads mapping fold coverage was calculated using qualimap_v2.040. We aimed to get a fold coverage of at least 50X for each genome, which was shown to be adequate for characterization of genomes41.…”

Investigation of the Evolutionary Development of the Genus Bifidobacterium by Comparative Genomics

“…However, it is worth mentioning that our in silico analyses are based on genome sequences that are still fragmented in a varying number of contigs (ranging from 1 to 56 contigs), having been assembled from a sequence coverage ranging from 45-to 281-fold. Nevertheless, a 50-fold sequence coverage of prokaryotic genomes, as obtained from next-generation sequencing (NGS) platforms, is predicted to represent more than 99% of the complete genome sequence (42), which implies an adequate reliability of the in silico analyses of ANI. Furthermore, it has been shown that for highly related genomes (i.e., ANI values of Ͼ94%), draft genome sequences that reach at least 50% of genome coverage for both strains result in ANI values that have been recognized as adequate for the calculation of nucleotide identity (28).…”

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“…For an extensive literature on assemblers consult [54,[69][70][71][72][73]. The list of aligners is updated online [53].…”