title: An integrated pipeline for next-generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899 (Digenea: Fasciolidae)
design: ~16 million 100 base-paired end reads were available for F. buski as a part of an independent attempt towards whole genome sequencing of F. buski. In order to recover mtDNA coding sequences from this data, Fasciola hepatica mt genome with accession AF216697.1 was retrieved from GenBank as a reference mt Genome and alignment using Bowtie (v2-2.0.0-beta6/bowtie2 --end-to-end --very-sensitive --no-mixed --phred64) [14]. In all, 1625 paired end reads were obtained, which were aligned to different intervals in the F. hepatica mt genome, covering ~ 3 kb of the 14 kb F. hepatica mt genome. Accordingly, primers were designed at these regions, using sequence information from F. buski to ensure optimum primer designing as shown in Table 1. Long-range PCR was carried out using 10 ng of genomic DNA from F. buski and the following PCR conditions: 10 ng of FD-2 DNA with 10 uM Primer mix in 10 ul reaction PCR cycling conditions – 98° C for 3min, 35 cycles of 98° C for 30 sec, 60 for 30 sec, 72 for 2 min 30sec, final extension 72° C for 3 min and 4° C hold. The bands were gel-eluted corresponding to different products and pooled for NGS library construction. The pooled PCR products were sheared to smaller sizes using Bioruptor. One each of Ion Torrent and Illumina library was constructed as per manufacturers’ protocols. Briefly, PCR products were sonicated, adapter ligated and amplified for x cycles to generate a library. The libraries were sequenced to generate 14k reads of an average of 150 nt SE reads on Ion Torrent, and 1.3 million reads of 72 nt SE reads on Illumina GAIIx. High quality and vector filtered reads from Ion Torrent and Illumina sequencing were assembled (hybrid-assembly) using Mira-3.9.15 (http://sourceforge.net/apps/mediawiki/mira-assembler).  The hybrid assembly generated 776 contigs.  All 776 contigs were then used as input for CAP3 assembler which generated 38 contigs.  The contigs were further filtered to remove short and duplicate contigs.