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Abstract
The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.
Highlights
The advent of next-generation sequencing technologies, combined with major advances in molecular and bioinformatics techniques, have enabled rapid growth in the culture-independent sequencing of amplified marker genes from environmental microbial communities
We present an alternative technique that takes advantage of a high-speed Burrows-Wheeler alignment procedure combined with rapid filtering and parsing of the data to remove bottlenecks in the pipeline
We achieve an order-of-magnitude speedup over conventional techniques without sacrificing accuracy or memory use, and in some cases improving both significantly
Summary
The advent of next-generation sequencing technologies, combined with major advances in molecular and bioinformatics techniques, have enabled rapid growth in the culture-independent sequencing of amplified marker genes (amplicons) from environmental microbial communities. The major benefit of amplicon sequencing is that it allows reasonable resolution of taxonomic composition in these communities at a fraction of the cost of deep metagenomic sequencing. Once these sequences are generated, a common analysis approach is to bin them by sequence identity into operational taxonomic units (OTUs)[1,2,3,4]. In habitats with mostly well-characterized microbes, we have the option of matching the generated amplicon sequences to reference databases containing example marker genes from known taxa [5]. NINJA-OPS, our portable, opensource OTU picking pipeline, realizes these goals
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