Mitogenome assembly from genomic multiplex libraries: comparison of strategies and novel mitogenomes for five species of frogs

Abstract

Next-generation sequencing continues to revolutionize biodiversity studies by generating unprecedented amounts of DNA sequence data for comparative genomic analysis. However, these data are produced as millions or billions of short reads of variable quality that cannot be directly applied in comparative analyses, creating a demand for methods to facilitate assembly. We optimized an in silico strategy to efficiently reconstruct high-quality mitochondrial genomes directly from genomic reads. We tested this strategy using sequences from five species of frogs: Hylodes meridionalis (Hylodidae), Hyloxalus yasuni (Dendrobatidae), Pristimantis fenestratus (Craugastoridae), and Melanophryniscus simplex and Rhinella sp. (Bufonidae). These are the first mitogenomes published for these species, the genera Hylodes, Hyloxalus, Pristimantis, Melanophryniscus and Rhinella, and the families Craugastoridae and Hylodidae. Sequences were generated using only half of one lane of a standard Illumina HiqSeq 2000 flow cell, resulting in fewer than eight million reads. We analysed the reads of Hylodes meridionalis using three different assembly strategies: (1) reference-based (using bowtie2); (2) de novo (using abyss, soapdenovo2 and velvet); and (3) baiting and iterative mapping (using mira and mitobim). Mitogenomes were assembled exclusively with strategy 3, which we employed to assemble the remaining mitogenomes. Annotations were performed with mitos and confirmed by comparison with published amphibian mitochondria. In most cases, we recovered all 13 coding genes, 22 tRNAs, and two ribosomal subunit genes, with minor gene rearrangements. Our results show that few raw reads can be sufficient to generate high-quality scaffolds, making any Illumina machine run using genomic multiplex libraries a potential source of data for organelle assemblies as by-catch.

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

“…The classic method is a time consuming and resource demanding task (Hahn, Bachmann & Chevreux, 2013). Recently, the developments of next generation sequencing (NGS) have brought about PCR-free high-throughput sequencing to recover the whole mtDNA genomes (Machado, Lyra & Grant, 2015; Gan, Schultz & Austin, 2014; Hahn, Bachmann & Chevreux, 2013; Jex et al, 2010). The NGS method overcomes some of the current challenges to isolate mtDNA and the biases introduced by PCR (Smith, 2016; Dames, Eilbeck & Mao, 2015; Machado, Lyra & Grant, 2015; Tang et al, 2014; Hahn, Bachmann & Chevreux, 2013).…”

“…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). …”

“…However, these rearranged sequences have been approached using the conventional procedure of combining long-range PCR with subsequent primer walking. Although high-quality mitochondrial genomes have been obtained from NGS for anurans (Machado, Lyra & Grant, 2015), more cases are needed to confirm the effectiveness of this protocol, especially for species with gene order rearrangement.…”

The mitochondrial genomes of three species of poison frogs (Anura: Dendrobates)

“…The assembly was carried out with MIRA v4.0 (Chevreux et al 1999) and MITObim v1.8 (Hahn et al 2013), following Machado et al (2016) and using the reference mitogenome of Bufo gargarizans (NC020048) as initial seed. Assemblies were manually verified in Geneious software v.6 (Biomatters, San Francisco, CA) to evaluate the coverage and quality of each mitochondrial element.…”

“…New sequences were submitted to GenBank (accession numbers MF069434-MF069441). Sequences were aligned to published complete or near-complete mitochondrial genomes of dendrobatid and aromobatid frogs (Zhang et al 2013;Machado et al 2016;Vacher et al 2016) with MAFFT v.7 (Katoh and Standley 2013). The mitogenome sequence of Bufo tibetanus was used as outgroup.…”