SOAPBarcode: revealing arthropod biodiversity through assembly of Illumina shotgun sequences of PCR amplicons

Abstract

Summary1. Metabarcoding of mixed arthropod samples for biodiversity assessment has mostly been carried out on the 454 GS FLX sequencer (Roche, Branford, Connecticut, USA), due to its ability to produce long reads (≥400 bp) that are believed to allow higher taxonomic resolution. The Illumina sequencing platforms, with their much higher throughputs, could potentially reduce sequencing costs and improve sequence quality, but the associated shorter read length (typically <150 bp) has deterred their usage in next-generation-sequencing (NGS)-based analyses of eukaryotic biodiversity, which often utilize standard barcode markers (e.g. COI, rbcL, matK, ITS) that are hundreds of nucleotides long. 2. We present a new Illumina-based pipeline to recover full-length COI barcodes from mixed arthropod samples. Our new assembly program, SOAPBarcode, a variant of the genome assembly program SOAPdenovo, uses paired-end reads of the standard COI barcode region as anchors to extract the correct pathways (sequences) out of otherwise chaotic 'de Bruijn graphs', which are caused by the presence of large numbers of COI homologs of high sequence similarity. 3. Two bulk insect samples of known species composition have been analysed in a recently published 454 metabarcoding study (Yu et al. 2012) and are re-analysed by our analysis pipeline. Compared to the results of Roche 454 (c. 400-bp reads), our pipeline recovered full-length COI barcodes (658 bp) and 17-31% more species-level operational taxonomic units (OTUs) from bulk insect samples, with fewer untraceable (novel) OTUs. On the other hand, our PCR-based pipeline also revealed higher rates of contamination across samples, due to the Illumina's increased sequencing depth. On balance, the assembled full-length barcodes and increased OTU recovery rates resulted in more resolved taxonomic assignments and more accurate beta diversity estimation. 4. The HiSeq 2000 and the SOAPBarcode pipeline together can achieve more accurate biodiversity assessment at a much reduced sequencing cost in metabarcoding analyses. However, greater precaution is needed to prevent cross-sample contamination during field preparation and laboratory operation because of greater ability to detect non-target DNA amplicons present in low-copy numbers.

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“…However, due to the significant decrease in quality as the individual reads exceed 200 nucleotides, the maximum length of sequence that can be generated with an error rate low enough to enable accurate identification is approximately 450 nucleotides (2 × 300 nucleotides paired reads on a MiSeq). Whilst the methodology is well established for bacterial communities sequencing a 250 nucleotide fragment of the v4 region of the 16S rRNA gene (Kozich et al 2013), this is considerably shorter than the standard barcode length for invertebrates of 650 bp, and in some applications the reduction in length results in poor species-level resolution (Liu et al 2013). Some researchers have explored the use of shorter regions of the standard barcode, or study alternate genes that may provide improved species resolution with shorter fragments; however, this presents the perennial issue of lack of reference sequences (Deagle et al 2014).…”

“…Some researchers have explored the use of shorter regions of the standard barcode, or study alternate genes that may provide improved species resolution with shorter fragments; however, this presents the perennial issue of lack of reference sequences (Deagle et al 2014). The large number of sequencing reads generated enables some solutions; the use of multiple, shorter overlapping amplicons to assemble longer amplicons or amplifying the standard COI barcode and producing a barcode sequence by assembling together sequences from each end with shotgun sequence of the whole amplicon (Liu et al 2013). Next-generation sequencing technologies are rapidly evolving (e.g., MinIon from Oxford Nanopore), and it is probable that within a short period of time they will be able to generate suitable read-lengths for DNA barcoding, enabling the use of existing databases.…”

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“…Although NGS can produce sequences more cheaply than Sanger sequencing, current NGS sequence read lengths are typically 200-400 bp, shorter than the standard 658 bp DNA barcoding region, limiting their utility for species-resolution identifications (Liu et al 2013). This limitation is being overcome as newly available platforms employing single molecule real time (SMRT) sequencing technology now yield read lengths of 3000-20 000 bp (Roberts et al 2013), covering the entire DNA barcoding region.…”

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“…The task of collating COI genotypes across all metazoans (and particularly small and cryptic invertebrates in remote regions) is arguably more difficult than creating reference data for the overall fewer conserved 18S rDNA genotypes. Critically, the remedy for this situation is to increase α taxonomic [78,79] approaches in the Antarctic region linked to sequencing efforts, the latter in the future being likely realized using shotgun sequencing approaches [16,58,80].…”