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

Abstract

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don’t contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.

The mitochondrial genome ofParagonimus westermani(Kerbert, 1878), the Indian isolate of the lung fluke representative of the family Paragonimidae (Trematoda)

“…2 and Table 2). The gene arrangement pact of protein-coding genes in P. westermani tallies with that of Fasciola hepatica (Le et al, 2000; Le, Blair & McManus, 2001), Opisthorchis felineus (Shekhovtsov et al, 2010), Fasciola gigantica (Liu et al, 2014), Fasciolopsis buski (Biswal et al, 2013) and Paramphistomum cervi (Yan et al, 2013) mt genomes, but is different from that seen in Taenia and Ascaris species (Nakao, Sako & Ito, 2003; Okimoto, Macfarlane & Wolstenholme, 1990) (Fig. 3).…”

“…2X KapaHiFiHotstart PCR ready mix (Kapa Biosystems Inc., Woburn, MA) reagent was used for PCR. The Ion Torrent library was made using Ion Plus Fragment library preparation kit (Life Technologies, Carlsbad, US) and the Illumina library was constructed using TruSeqTM DNA Sample Preparation Kit (Illumina, Inc., US) reagents for library prep and TruSeq PE Cluster kit v2 along withTruSeq SBS kit v5 36 cycle sequencing kit (Illumina, Inc., US) for sequencing (Biswal et al, 2013). PCR products were sonicated, adapter ligated and amplified for x cycles to generate a library and subsequently were sequenced to generate reads of an average of 121 nt SE reads on Ion Torrent.…”

Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearranged mitochondrial genomes inAmolops chunganensisandQuasipaa boulengeri

“…Based on high-throughput sequencing technologies, Tang et al (2014) developed a novel multiplex sequencing and assembly pipeline for rapid and accurate reconstruction of full mitogenome from pooled Drosophila without DNA enrichment or amplification. The fast recovery, assembly, and annotation of mitogenome from genomic sequencing have been applied in butterflies and moths (Cong & Grishin, 2016), crayfish (Gan, Schultz & Austin, 2014), monogenean ectoparasitic flat-worms (Hahn, Bachmann & Chevreux, 2013), giant intestinal fluke ( Fasciolopsis buski ; Biswal et al, 2013) and Ascidian species (Rubinstein et al, 2013). In addition, RNA-seq and ultraconserved elements (UCE) sequencing are also excellent source to assemble mitochondrial genomes (Machado, Lyra & Grant, 2015; Moreira, Furtado & Parente, 2015; Raposo do Amaral et al, 2015).…”

Complete Taiwanese Macaque (Macaca cyclopis) Mitochondrial Genome: Reference-Assisted de novo Assembly with Multiple k-mer Strategy

“…Initially, the most common approach used multiplex PCR to capture overlapping fragments that could be assembled into longer sequence [ 4 , 7 , 9 , 10 ]. Later, amplification of longer fragments by long-range PCR or multiplex PCR [ 8 ] and NGS [ 20 – 22 ] was employed for assembly.…”