Updating benchtop sequencing performance comparison

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

Whole-genome sequencing is becoming commonplace, but the accuracy and completeness of variant calling by the most widely used platforms from Illumina and Complete Genomics have not been reported. Here we sequenced the genome of an individual with both technologies to a high average coverage of ∼76×, and compared their performance with respect to sequence coverage and calling of single-nucleotide variants (SNVs), insertions and deletions (indels). Although 88.1% of the ∼3.7 million unique SNVs were concordant between platforms, there were tens of thousands of platform-specific calls located in genes and other genomic regions. In contrast, 26.5% of indels were concordant between platforms. Target enrichment validated 92.7% of the concordant SNVs, whereas validation by genotyping array revealed a sensitivity of 99.3%. The validation experiments also suggested that >60% of the platform-specific variants were indeed present in the genome. Our results have important implications for understanding the accuracy and completeness of the genome sequencing platforms.

Background De Bruijn graphs are a theoretical framework underlying several modern genome assembly programs, especially those that deal with very short reads. We describe an application of de Bruijn graphs to analyze the global repeat structure of prokaryotic genomes. Results We provide the first survey of the repeat structure of a large number of genomes. The analysis gives an upper-bound on the performance of genome assemblers for de novo reconstruction of genomes across a wide range of read lengths. Further, we demonstrate that the majority of genes in prokaryotic genomes can be reconstructed uniquely using very short reads even if the genomes themselves cannot. The non-reconstructible genes are overwhelmingly related to mobile elements (transposons, IS elements, and prophages). Conclusions Our results improve upon previous studies on the feasibility of assembly with short reads and provide a comprehensive benchmark against which to compare the performance of the short-read assemblers currently being developed.