An Integrated Pipeline for de Novo Assembly of Microbial Genomes

Abstract

Remarkable advances in DNA sequencing technology have created a need for de novo genome assembly methods tailored to work with the new sequencing data types. Many such methods have been published in recent years, but assembling raw sequence data to obtain a draft genome has remained a complex, multi-step process, involving several stages of sequence data cleaning, error correction, assembly, and quality control. Successful application of these steps usually requires intimate knowledge of a diverse set of algorithms and software. We present an assembly pipeline called A5 (Andrew And Aaron's Awesome Assembly pipeline) that simplifies the entire genome assembly process by automating these stages, by integrating several previously published algorithms with new algorithms for quality control and automated assembly parameter selection. We demonstrate that A5 can produce assemblies of quality comparable to a leading assembly algorithm, SOAPdenovo, without any prior knowledge of the particular genome being assembled and without the extensive parameter tuning required by the other assembly algorithm. In particular, the assemblies produced by A5 exhibit 50% or more reduction in broken protein coding sequences relative to SOAPdenovo assemblies. The A5 pipeline can also assemble Illumina sequence data from libraries constructed by the Nextera (transposon-catalyzed) protocol, which have markedly different characteristics to mechanically sheared libraries. Finally, A5 has modest compute requirements, and can assemble a typical bacterial genome on current desktop or laptop computer hardware in under two hours, depending on depth of coverage.

Whole-Genome Sequencing of the Mycobacterium tuberculosis Manila Sublineage Results in Less Clustering and Better Resolution than Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Typing and Spoligotyping

“…In order to detect polymorphisms in regions not present in the H37Rv reference strain, we constructed a custom-made reference sequence. Briefly, the reference strain for polymorphism calling was custom made by joining the full sequence of M. tuberculosis reference strain H37Rv (GenBank accession number NC_018143.1), and contigs were de novo assembled using the A5 pipeline (14) from all short reads of a randomly selected Manila isolate that did not align to the H37Rv reference strain, as determined by the Mosaik assembler (https://code.google.com/p/mosaik-aligner/). These contigs were concatenated after the H37Rv sequence using the string NNNNNCATTCCATTCATTAATTAATTAATGAAT GAATGNNNNN as a separator.…”

Characterization of Invasive Group B Streptococcus Strains from the Greater Toronto Area, Canada

“…Polymorphisms were called against the reference pseudochromosomes using a variant ascertainment algorithm (VAAL) (31). The A5 pipeline was used for de novo assembly of newly sequenced GBS strains (32). Contigs Ͼ100 nucleotides in length were then used to search the NCBI nonredundant database using BLAST (33).…”

Deriving Group A Streptococcus Typing Information from Short-Read Whole-Genome Sequencing Data

“…The multiplexed sequencing reads were parsed and barcode information was removed using onboard software. The A5 pipeline was used for de novo assembly of newly sequenced GAS strains (27). The trimmed and untrimmed emm type reference databases were downloaded directly from CDC ftp sites (ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm and ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/emmsequ, respectively).…”