Submitted 8 August 2016Accepted 11 November 2016Published 15 December 2016

Corresponding authorYun Xia xiayuncibaccn

Academic editorKorbinian Schneeberger

Additional Information andDeclarations can be found onpage 11

DOI 107717peerj2786

Copyright2016 Yuan et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Next-generation sequencing of mixedgenomic DNA allows efficient assemblyof rearranged mitochondrial genomesin Amolops chunganensis and QuasipaaboulengeriSiqi Yuan12 Yun Xia1 Yuchi Zheng1 and Xiaomao Zeng1

1Department of Herpetology Chengdu Institute of Biology Chinese Academy of Sciences Chengdu SichuanChina

2University of Chinese Academy of Sciences Beijing China

ABSTRACTRecent improvements in next-generation sequencing (NGS) technologies can facilitatethe obtainment of mitochondrial genomes However it is not clear whether NGS couldbe effectively used to reconstruct the mitogenome with high gene rearrangementThese high rearrangements would cause amplification failure andor assembly andalignment errors Here we choose two frogs with rearranged gene order Amolopschunganensis and Quasipaa boulengeri to test whether gene rearrangements affectthe mitogenome assembly and alignment by using NGS The mitogenomes withgene rearrangements are sequenced through Illumina MiSeq genomic sequencing andassembled effectively by Trinity v210 and SOAPdenovo2 Gene order and contents inthe mitogenome of A chunganensis andQ boulengeri are typical neobatrachian patternexcept for rearrangements at the position of lsquolsquoWANCYrsquorsquo tRNA genes cluster Furtherthe mitogenome of Q boulengeri is characterized with a tandem duplication of trnMMoreover we utilize 13 protein-coding genes of A chunganensis Q boulengeri andother neobatrachians to reconstruct the phylogenetic tree for evaluating mitochondrialsequence authenticity of A chunganensis and Q boulengeri In this work we providenearly complete mitochondrial genomes of A chunganensis and Q boulengeri

Subjects Evolutionary Studies Genetics Genomics Molecular Biology ZoologyKeywords Gene rearrangement WANCY Illumina sequencing Neobatrachian Mitogenome

INTRODUCTIONIn metazoan mitochondrial genomes the organization is usually conserved (Boore 1999)The typical mitogenome of metazoans contains two ribosomal RNAs 22 transfer RNAs(tRNAs) 13 protein-coding genes (PCGs) and non-genic regions Because of maternalinheritance features and other characteristics of mitogenome (ie relatively conserved genecontent and organization rapid mutation rate and limited recombination) mitochondrialDNA (mtDNA) is a valuable and popular molecular marker (Xia et al 2014 Hahn Bach-mann amp Chevreux 2013 Zhang et al 2013) It has been extensively applied in populationgenetics evolutionary biology phylogeography as well as phylogenetic relationships

How to cite this article Yuan et al (2016) Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearrangedmitochondrial genomes in Amolops chunganensis and Quasipaa boulengeri PeerJ 4e2786 DOI 107717peerj2786

at multiple taxonomic levels (Hahn Bachmann amp Chevreux 2013 Zhang et al 2013Lindqvist et al 2010) However cases of mitochondrial reorganization (eg gene rear-rangements gene duplication and loss) are found in many lineages even in closely-relatedspecies (Xia et al 2014 Kurabayashi et al 2010 Dowton et al 2009) The rearrangementswould cause amplification assembly and alignment errors or failure

The traditional protocols to produce themitochondrial genomes have relied on perform-ing either standardlong PCRor cloning followed by a series of Sanger sequencing (MoreiraFurtado amp Parente 2015 Gan Schultz amp Austin 2014 Poulsen et al 2013) The classicmethod is a time consuming and resource demanding task (Hahn Bachmann amp Chevreux2013) Recently the developments of next generation sequencing (NGS) have broughtabout PCR-free high-throughput sequencing to recover the whole mtDNA genomes(Machado Lyra amp Grant 2015Gan Schultz amp Austin 2014Hahn Bachmann amp Chevreux2013 Jex et al 2010) The NGSmethod overcomes some of the current challenges to isolatemtDNA and the biases introduced by PCR (Smith 2016 Dames Eilbeck amp Mao 2015Machado Lyra amp Grant 2015 Tang et al 2014 Hahn Bachmann amp Chevreux 2013)

Numerous applications for rapidly assembling mitogenomes directly from NGS havebeen proposed (Cong amp Grishin 2016 Lounsberry et al 2015 Cameron 2014 Gan Schultzamp Austin 2014) Based on high-throughput sequencing technologies Tang et al (2014)developed a novel multiplex sequencing and assembly pipeline for rapid and accuratereconstruction of full mitogenome from pooled Drosophila without DNA enrichment oramplification The fast recovery assembly and annotation of mitogenome from genomicsequencing have been applied in butterflies and moths (Cong amp Grishin 2016) crayfish(Gan Schultz amp Austin 2014) monogenean ectoparasitic flat-worms (Hahn Bachmann ampChevreux 2013) giant intestinal fluke (Fasciolopsis buski Biswal et al 2013) and Ascidianspecies (Rubinstein et al 2013) In addition RNA-seq and ultraconserved elements (UCE)sequencing are also excellent source to assemble mitochondrial genomes (Machado Lyraamp Grant 2015 Moreira Furtado amp Parente 2015 Raposo do Amaral et al 2015)

The recent availability of complete mitogenomes and the partial sequences of lsquolsquohotspotsrsquorsquoofmtDNArearrangements revealed a surprising array of gene organization in anurans espe-cially in neobatrachians (Xia et al 2014Kurabayashi et al 2010Ren et al 2009) Gene or-der rearrangement involving the coding geneND5 is observed inDicroglossidae (EuphlyctisHoplobatrachus Fejervarya Alam et al 2010 Ren et al 2009 Liu Wang amp Su 2005)Mantellidae (Mantella Kurabayashi et al 2008 Kurabayashi et al 2006) and Rhacophori-dae (Rhacophorus Buergeria Huang et al 2016 Sano et al 2005 Sano et al 2004) Twocopies of trnM genes are found in Dicroglossidae (eg Feirana and Quasipaa Chen et al2015 Shan et al 2014 Ren et al 2009 Liu Wang amp Su 2005) and Mantellidae (egMan-tellaKurabayashi et al 2008Kurabayashi et al 2006) The tRNAgene cluster lsquolsquoWANCYrsquorsquois a hotspot of gene rearrangement in Neobatrachia such as Ranidae (Xia et al 2014Kurabayashi et al 2010) Dicroglossidae (Shan et al 2014 Zhou et al 2009) and Mantel-lidae (Kurabayashi et al 2008 Kurabayashi et al 2006) However these rearranged se-quences have been approached using the conventional procedure of combining long-rangePCR with subsequent primer walking Although high-quality mitochondrial genomeshave been obtained from NGS for anurans (Machado Lyra amp Grant 2015) more cases are

Yuan et al (2016) PeerJ DOI 107717peerj2786 218

needed to confirm the effectiveness of this protocol especially for species with gene orderrearrangement

Some frogs with gene order rearrangements provide the opportunity to test the validity ofmitogenome assembly by NGS Gene order reorganization was reported in the lsquolsquoWANCYrsquorsquogene cluster of some Amolops mitogenomes (Xia et al 2014 Xue et al 2015 Zhang Xiaamp Zeng 2016 Shan et al 2016) The lsquolsquoWANCYrsquorsquo gene cluster was also rearranged in thespiny-bellied frog Quasipaa boulengeri (Shan et al 2014) Furthermore the intraspecificdiversity of gene rearrangements which contains four kinds of rearrangements withinQ boulengeri has been identified in 290 samples from 28 populations (Xia et al 2016)The discovery of intermediate states and alternative losses types in the spiny-bellied frogprovided direct evidence of tandem duplication and random loss model for mitochondrialgene rearrangements However such partial duplications and deletions of mtDNAfragments would cause inconvenience to assemble mitogenomes by NGS

In this study we choose two frogs with gene rearrangements Amolops chunganensis andQuasipaa boulengeri to testwhetherNGS could effectively obtain themitogenomewith highgene rearrangement The mitogenomes are assembled and annotated from next generationsequencing reads through Illumina MiSeq genomic sequencing The nearly-completemitochondrial DNA sequence of A chunganensis and Q boulengeri were recovered andcompared with otherAmolops andQuasipaa species respectively In order to evaluatemito-chondrial sequence authenticity of A chunganensis andQ boulengeri we performed a phy-logenetic analysis The phylogenetic tree was constructed by using Bayesian Inference (BI)and Maximum Likelihood (ML) methods for the two newly obtained and other publishedneobatrachian mitogenomes from GenBank

MATERIALS AND METHODSLibrary preparation and Illumina sequencingThe sample of Amolops chunganensiswas collected in Gansu Province China (Voucher NoXM5526 ) and Quasipaa boulengeri was collected in Sichuan Province China (VoucherNo XM3632 ) Chengdu Institute of Biology issued permit number CIB2014-36 andCIB2014-110 for field work All work with animals was conducted according to relevantnational and international guide-lines on the Protection of Wildlife All animal care andexperimental procedures were approved by the Animal Care and Use Committee (PermitNumber CIB-20121220A) Chengdu Institute of Biology Chinese Academy of Sciences

Genomic DNA was extracted from muscle tissue through SDS-proteinase Kphenol-chloroform protocols DNA samples were shipped toNovogene Bioinformatics Technology(Beijing China) for library construction and sequencing on Illumina MiSeq platform with300 bp paired-end reads (PE300) Briefly the paired-end librarywas performedwith TruSeqkit (insert size sim500 bp) following the protocols of Illumina DNA sample preparationThese libraries were pooled together to sequence on Illumina MiSeq platform at theNovogene Bioinformatics Technology (Beijing China) Sequence files for genomic DNAfrom A chunganensis and Q boulengeri were generated in FASTQ format (sequence readand quality information in Phred format) The raw sequencing data were deposited in

Yuan et al (2016) PeerJ DOI 107717peerj2786 318

the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRAhttpwwwncbinlmnihgovTracessra) under accession numbers SRR3929655 andSRR3929656 for Q boulengeri and A chunganensis respectively

Mitogenome assemblyContaminant sequences were first removed Then low-quality regions (Phred qualityscore lt 20) and sequence adapters in the libraries were trimmed by using Trimmomatic(Bolger Lohse amp Usadel 2014) De novo assembly of clean reads for each datasets wereperformed using SOAPdenovo2 (Luo et al 2012) and Trinity v210 (Haas et al 2013)Reads preprocess and assembly parameters followed the individual program guidelines ForSOAPdenovo2 we used 7 k-mer sizes between 50 and 80 with a step size of 5 and manuallymodified the assembly parameter values Trinity assembler was used with the inchwormk-mer method following authorsrsquo recommendations For each assembly we monitoredthe change of total number of contigs and N50 size over the assessed parameter range

After assembly we used the published mitogenomes of Amolops as queries againstthe contigs dataset of A chunganensis Amolops loloensis (GeneBank No KT750963) andA mantzorum (KJ546429)were used as queries for the referencemitogenome The referencemitogenomes were BLASTed against assembly using BLASTn (BLAST+ v2230) to searchfor contigs with mitochondrial protein-coding and RNA genes ForQ boulengeri the samestrategy has been performedQuasipaa boulengeri (KC686711) andQ spinosa (NC_013270)were chose as reference mitogenomes

Mitogenome annotation and analysisThe mitogenome sequence was annotated using both tRNAscan-SE v121 (httplowelabucscedutRNAscan-SE Schattner Brooks amp Lowe 2005 Lowe amp Eddy 1997) and the MI-TOS web server (httpmitosbioinfuni-leipzigdeindexpy Bernt et al 2013) These pro-grams also used to predict the potential cloverleaf secondary structures of tRNAs genes Thesequences of candidate tRNAs were recognized and aligned with homologous tRNAs genesThirteen PCGs of the twomitogenomeswere identified by comparing inferred open readingframes with published Amolops and Quasipaa species Both translation of open readingframes and annotation of the N- and C- terminal ends of each PCG were done in MEGAv606 (Tamura et al 2013) In addition the boundaries of the rrnL and rrnS genes werepredicted by the flanking tRNA genes

Phylogenetic analysisTo validate the phylogenetic relationship of A chunganensis and Q boulengeri amongneobatrachians we aligned 46 mitogenomes of neobatrachians to confirm the phylogeneticrelationships and selected Pelobates cultripes (NC_008144) Xenopus laevis (NC_001573)and X tropicalis (NC_006839) as outgroup taxa Details of the GenBank accession numbersof anuran mitochondrial genomes analyzed were listed in Table S1 All sequences wereedited manually in BioEdit Sequence Alignment Editor v705 (Hall 1999) and MEGAv606 (Tamura et al 2013) with default parameters The alignments of 13 PCGs weredetermined in software MEGA v606 with the default settings (align codons) The PCGs

Yuan et al (2016) PeerJ DOI 107717peerj2786 418

were translated to amino acids sequences and were manually concatenated all sequencesinto a single nucleotide dataset (total 11502 bp)

We constructed the phylogenies with the concatenated dataset using BI andMLmethodswhich were conducted by MrBayes v31 (Ronquist amp Huelsenbeck 2003 Huelsenbeck ampRonquist 2001) and RAxML v82x (Stamatakis 2014) respectively Nucleotide substitu-tion model selection was estimated under Bayesian Information Criterion (BIC) scores injModeltest v085 (Posada 2008) and model GTR + I + G was selected as the best-fittingmodel for BI andML analyses BI partitioning analysis followed the programs with calculat-ing amajority rule consensus treewith 10000000 generations ofMarkov chainMonteCarlo(MCMC) with frequency of tree sampling every 1000 generations and the first 25000 treesdiscarding as burn-in and starting from a random tree After performing two independentruns the output trees were combined to estimate the Bayesian posterior probabilities (BPP)in 50 majority rule for each node For ML analysis program RAxML were performedwith model GTRGAMMA under the similar partitioning parameters as BI analysis andwith 1000 bootstrap replicates to calculate the bootstrap (BS) of the topology In additionthe significant nodesrsquo supports were considered with 95 BPP (Felsenstein 2004) and 75BS (Hillis amp Bull 1993) in BI and ML analysis respectively

RESULTSSequencing data from Illumina MiSeqA total of 1529million rawPE readswere produced on IlluminaMiSeq (PE300) systems (49Gb and 42 Gb raw data for Q boulengeri and A chunganensis respectively) After removalof adaptors and low-quality reads the clean reads were used for the subsequent assemblingAll assembly statistics of each assembler were shown in Table 1 Although total numbers ofassembled contigs in Trinity were less than SOAPdenovo2 the Trinity assembler collectedlarger N50 and longer contigs (Table 1) More than two hundred contigs were alignedto reference mitogenome for each K-mer assembly of SOAP denovo2 however most ofthem less than 500 bp Further few gaps existed when such contigs were used to assemblefor the final mitogenome construction By contrast the best aligned contigs to referencemitogenome in Trinity are longer than 16 kb without gaps It implies that Trinity is betterthan SOAPdenovo2 to recover mitogenome for low coverage metagenomic skimming data

Mitogenomic sequences and annotationWe recovered the nearly complete mitogenome of two species A chunganensis andQ boulengeri For the mitochondrial genome of A chunganensis this is the first workreporting the nearly complete mitochondrial genome The nearly complete mitogenomesequences of A chunganensis and Q boulengeri were 16795 bp and 16672 bp in totallength respectively (GenBank accession no KX645666 and KX645665) The mitogenomeof A chunganensis contained 22 transfer RNAs 2 ribosomal RNAs 13 PCGs and partialD-loop region (Table S2) Comparatively the mitogenome of Q boulengeri contained 23transfer RNAs with a tandem duplication of trnM (Table S3) The annotation and genemap of two mitogenomes were illustrated in Fig 1 In these mitogenomes eight tRNAsand nad6 gene were encoded in the L-strand and the rest of genes were encoded in the

Yuan et al (2016) PeerJ DOI 107717peerj2786 518

Table 1 Statistics and assembled result of SOAP denovo2 and Trinity

Species Assembly method Total number of contigs Total number of length N50 Max contig length

K50a 6341679 1860878175 300 2615K55a 6086855 1875157178 314 2880K60a 5715340 1870183020 332 3183K65a 5460129 1843817246 338 2817K70a 5122048 1779429007 332 2980K75a 4926588 1723696265 318 2779K80a 4686214 1630358728 300 2830

Quasipaaboulengeri

Trinity 1555983 755862296 526 16672K50a 5072290 1417075271 300 2537K55a 4955300 1436818547 300 2741K60a 4746605 1436171325 300 2416K65a 4549311 1407207578 300 2513K70a 4301826 1350614260 300 2527K75a 4167451 1308301284 300 2578K80a 4007361 1244363080 300 4366

Amolopschunganensis

Trinity 1017500 448075968 469 16795

NotesaThe k-mer sizes used in SOAP denovo2

L1 T F

P

rrnLrrnS V L2 ND1 ND2 ND3 ND4 ND5

ND6

COI COII COIII cytbATP8 ATP6 ND4LI M1

Q

W

A N C Y

D

S2

KM2

E

H S1G R

B Quasipaa boulengeri

L1 T F

P

rrnLrrnS V L2 ND1 ND2 ND3 ND4 ND5

ND6

COI COII COIII cytbATP8 ATP6 ND4LI M

Q

W

ANCY

D

S2

Krsquo

E

G R H S1

A Amolops chunganensis

K

Figure 1 Annotation andmap of Amolops chunganensis andQuasipaa boulengeri Annotation of Amolops chunganensis (A) and Quasipaaboulengeri (B) mitogenome PCGs are colored in red tRNA-coding genes are in blue rrnL and rrnS are in green A pseudogene of trnK is locatedbetween cox2 and atp8 genes Each gene is shown as an arrow indicating the transcription direction The arrows on top of the black line correspondto genes coded on the H-strand and those below show genes on the L-strand

H-strand (Fig 1 Tables S2 and S3) as described in other anurans (Shan et al 2016 Shanet al 2014 Zhang et al 2013) The tRNA-coding genes length ranged from 60 to 73 bpSecondary structures developed by MITOS suggested that all tRNAs fold in a cloverleafstructure (Figs S1 and S2)

In A chunganensis mitogenome ten of 13 PCGs started with the common initiationcodon ATG while nad2 began with ATT cox1 and atp6 initiated with ATA (Table S2)

Yuan et al (2016) PeerJ DOI 107717peerj2786 618

For the terminal codon atp8 cox1 and nad6 genes stop with TAG AGG and AGArespectively nad4l nad4 and cytb ended with TAA while nad2 and atp6 ended with TAFive PCGs (cox2 cox3 and nad1 nad3 nad5) ended with an incomplete stop codon(Tndash) In addition we had checked two repeated units 5prime-CCGTATGGTTTAATT -ATATACCATCTAATAGGTGATATATATACA-3prime (8 times) and 5prime-CCTATATATGCC-CGATATATACTATACTAAGTATTAATCA-3prime (6 times) located at the upstream of trnLand the downstream of cytb respectively Because of the tandem repeat units only 530 bphad been recovered in the D-loop region for A chunganensis

In Q boulengeri mitogenome eleven of 13 PCGs started with ATG and the nad2 andcox1 initiated with ATT and ATA respectively (Table S3) There were four kinds of stopcodon in the 13 PCGs AGG (cox1 and nad6) TAA (atp8 cytb and nad4l) TAG (nad5) andincomplete codonTndashin all other seven genes The incomplete stop codon (Tndash) is presumablycompleted as TAA by post transcriptional polyadenylation (Huang et al 2014 OjalaMontoya amp Attardi 1981) Overall nucleotide compositions of those mtDNA sequenceswere A (285) G (148) T (301) and C (265) in A chunganensis and A (278)G (148) T (283) and C (291) in Q boulengeri respectively Moreover we hadchecked one repeated unit 5prime-TTTTAAGTTA-3prime (25 times) located at the upstream of trnLSimilarly these multiple tandem repeated units caused the incomplete assembly of theD-loop region in Q boulengeri

Gene rearrangementThe gene order in A chunganensis and Q boulengeri follows the typical organization ofneobatrachians except some rearrangements of tRNA genes (Zhang et al 2005 Zhang etal 2013 Kurabayashi amp Sumida 2013) Compared with reported ranid frogs the locationof replication origin (OL) in A chunganensismitogenome was rearranged This gene orderwas consistent with other reported Amolops species (Zhang Xia amp Zeng 2016 Xue et al2015 Xia et al 2014) The OL is located in the tRNA genes cluster lsquolsquoWANCYrsquorsquo The typicalgene order of the lsquolsquoWANCYrsquorsquo cluster is trnW trnA trnN OL trnC and trnY

In A chunganensis however we found OL was located between the trnW and trnA It issimilar to A mantzorum and A loloensis gene arrangement pattern (Shan et al 2016 Xueet al 2015) Interestingly a trnK pseudogene was located between cox2 and atp8 genes (Fig1A Table S2) Compared with the trnK gene the pseudogene has a nucleotide deletion inthe anticodon loop and has some mutations in the acceptor stem of its secondary structure(Table S2 and Fig S1) Additionally theW-OL-ANCY structure from assembled NGS readswas consistent with previous reported gene rearrangement pattern of another individualof A chunganensis (KF771328 Xia et al 2014)

In the mitogenome of Q boulengeri two trnM genes were derived from a tandemduplication which was consistent with other Quasipaa species (Chen et al 2015 Shanet al 2014 Zhou et al 2009) Moreover pseudogene or residues of OL and trnN wereobserved in the tRNA genes cluster lsquolsquoWANCYrsquorsquo (between trnW and trnY ) resulting torearranged pattern lsquolsquoWAN-OL-Nrsquo-OLrsquo-CYrsquorsquo in this individual Furthermore the gene orderof the two available Q boulengeri individuals (GenBank no KF199152 KC686711 TableS1) were quite different in the lsquolsquoWANCYrsquorsquo region This indicated that the intraspecific

Yuan et al (2016) PeerJ DOI 107717peerj2786 718

diversity of gene rearrangements existed in Q boulengeri mitogenomes In addition576 non-coding nucleotides were observed between trnN and trnC (Fig 1B) which issimilar to other individuals of Q boulengeri by Sanger sequencing (Xia et al 2016) Theseobservations supported that mitogenome with gene order rearrangement can be obtainedeffectively and correctly from assembled NGS reads

Phylogenetic analysisThe concatenated PCGs data of mitogenome sequences contained 11502 nucleotidepositions including 3630 conserved sites 7806 variable sites and 7068 potentiallyparsimony-informative sites Bayesian inference and Maximum likelihood methods withGTR + I + G model consistently supported the same topology

The results of phylogenetic analysis revealed that monophyly of the Neobatrachia waswell supported by our work and previous molecular studies (Xia et al 2014 Roelants etal 2007 Frost et al 2006) The family Dicroglossidae was the sister clade to the clade((Rhacophoridae+Mantellidae)+ Ranidae) (Fig 2) In phylogenetic tree all Amolops andQuasipaa species formed a monophyly respectively The Amolops clade was split into twosub-clades One sub-clade containedA ricketti andA wuyiensis and the other one includedA chunganensis A loloensis A mantzorum and A tuberodepressus (Fig 2) The molecularphylogenetic result was consistent with the previous studies (Zhang Xia amp Zeng 2016 LvBi amp Fu 2014 Cai et al 2007) Three Q boulengeri individuals formed a monophyleticgroup sister toQ verrucospinosa CladeQuasipaa comprisedQ yei as the sister taxon to thesub-clade (100 100) containing (Q shini + ((Q jiulongensis + (Q spinosa + Q exil-ispinosa)) + (Q boulengeri + Q verrucospinosa))) (Fig 2) The phylogenetic relationshipssupported the authenticity of the two obtained mitogenomes among neobatrachians

DISCUSSIONEfficient assembly of mitochondrial genomesThe length of the best fitted contigs forA chunganensis andQ boulengeriwas 16795 bp and16672 bp respectively We failed to recover the complete mitogenome of A chunganensisand Q boulengeri due to the highly repetitive sequences in the D-loop region Except thegap in the D-loop region the whole mitogenomes were assembled successfully even ifthere are gene rearrangement in both species Repeated regions are a well-known problemfor sequence assembly algorithms and it was hard to assemble the D-loop region withextensive repeated regions by NGS (Hahn Bachmann amp Chevreux 2013 Tang et al 2014)In the D-loop region different repeated units repetitive sequence and repetitions resultedin different sequence length (length polymorphism) For example in Amolops species theD-loop region ranged from 2211 bp (A mantzorum) to 3391 bp (A loloensis) (ZhangXia amp Zeng 2016 Shan et al 2014 Xue et al 2015) Actually even if long tandem repeatregions can be successfully amplified by long PCR they also represent a problem for Sangersequencing (Xia et al 2014)

Gene order rearrangement is not a big problem for the assembly of mitogenomesby NGS In the assembly process the highly covered regions may be removed if uniformcoverage of reads are assumed by a de novo genome assembler (Rubinstein et al 2013) This

Yuan et al (2016) PeerJ DOI 107717peerj2786 818

100100

100100

100100

100100

100100

100100

100100

100100

100100

10010010099

100100

10056 100100

100100

100100100100

100100

10068

10010010082

100100

10082

100100

100100100100

100-

100100

100100

100100

100100

100100100100

100100

100100

100100

100100

100100

100100100100

100100

100100

098- 100100

100100

100100100100

100100

100100

KF199152 Quasipaa boulengeriKX645665 Quasipaa boulengeri

KF686711 Quasipaa boulengeri

NC_024843 Quasipaa yeiKF199148 Quasipaa shini

KF199147 Quasipaa verruspinosa

KF199149 Quasipaa jiulongensis

KF199151 Quasipaa exilispinosa

NC_013270 Quasipaa spinosa

NC_024180 Amolops mantzorum

KR559270 Amolops tuberodepressusNC_029250 Amolops loloensis

KX645666 Amolops chunganensis

NC_025591 Amolops wuyiensis

NC_023949 Amolops ricketti

KF199146 Nanarana taihangnica

NC_016119 Nanarana pleskei

NC_026789 Nanarana parkeri

AY899241 Limnonectes fragilisAY899242 Limnonectes bannaensis

NC_007440 Limnonectes fujianensis

KJ815050 Odorana margaretae

NC_009264 Pelophyla plancyiNC_002805 Pelophyla nigromaculata

NC_009423 Odorana tormotus

AB511282 Odorana ishikawae

NC_024548 Rana kunyuensis

NC_023528 Rana dybowskiiNC_023529 Rana chensinensis

AB511282 Rana catesbeiana

KF771342 Glandirana tientaiensisKF771341 Glandirana rugosa

NC_007888 Mantella madagascariensis

NC_008975 Buergeria buergeri

NC_007178 Rhacophorus schlegeliiNC_006405 Kaloula pulchra

NC_009422 Microhyla ornataNC_006403 Hyla chinensis

NC_010232 Hyla japonica

NC_009886 Bufo japonicusNC_008410 Bufo gargarizans

NC_008144 Pelobates cultripes

NC_001573 Xenopus laevis

NC_006839 Xenopus tropicalis

NC_014585 Euphlyctis hexadactylus

KC196066 Hoplobatrachus gugulosus

NC_012647 Fejervarya cancrivoraNC_029754 Fejervarya multistriataNC_005055 Fejervarya limnocharis

NC_014685 Occiozyga martensii

NC_014581 Hoplobatrachus tigerinus

04

Dicroglossidae

Ranidae

Mantellidae

Rhacophoridae

Microhylidae

Hylidae

Bufonidae

Pelobatidae

PipidaeOutgroups

NeobatrachiaAmolops

Quasipaa

Figure 2 The Bayesian Inference (BI) andMaximum Likelyhood (ML) tree (combined 13 PCGs 11502 bp) The Bayesian Inference (BI) andMaximum Likelihood (ML) tree based on nucleotide sequences of the combined 13 protein-coding genes (11502 in size) For the BI tree the GTR+ I+ G model was selected and two independent runs were performed for 1000000 generations with sampling frequency 0001 The GenBank ac-cession numbers of all species are shown Numbers beside the nodes are Bayesian posterior probabilities and ML Bootstrap respectively (showed inBPPBS) between cox2 and atp8 genes Each gene is shown as an arrow indicating the transcription direction The arrows on top of the black linecorrespond to genes coded on the H-strand and those below show genes on the L-strand

may be disadvantageous to the assembly of mitogenomes with gene order rearrangementTandem duplication followed by random loss (TDRL) is the most frequently invokedmodel to explain the diversity of gene rearrangements in metazoan mitogenomes (Boore2000) According to this model two copies of mitochondrial gene fragment would existafter tandem duplication These tandem duplications would cause errors when using shortreads of NGS to assemble mitogenomes Nevertheless deletion of a redundant gene-copymay happen rapidly due to replication andor transcription efficiency facilitating theformation of pseudogenes or the complete deletion of redundant genes Such pseudogenesor residues of tandem repeats could be efficiently distinguished from the original copy

Yuan et al (2016) PeerJ DOI 107717peerj2786 918

By aligning published mitogenomes of close species and individuals in rRNAs tRNAsand PCGs we assessed the quality of our assembled data The alignment results suggestedthat the assembled sequences were consistent with majority of other anurans mitogenomesThe non-conservative regions were located between nad2 and cox1 which includes generearrangements in Amolops and Quasipaa reported previously (Xia et al 2014 Shan et al2014 Shan et al 2016)

Our results illustrated that mitochondrial sequences can be successfully assembled fromthe NGS raw data Compared with traditional methods the strategy of mitogenomesassembly from NGS raw data has two distinct advantages First it neither depends onspecific primers nor be affected by gene rearrangement When gene rearrangement regioninclude primers the PCR-based method could not be successfully amplified Second thisapproach is fast timesaving relatively cheap and does not require great effort to recoverthe mitochondrial genomes (Gan Schultz amp Austin 2014 Tang et al 2014 Rubinstein etal 2013 Haiminen et al 2011) However the disadvantage of such an approach is that itrequires constructing separate genomic libraries for each sample

Mitochondrial gene order rearrangementsSome pseudogenes or residues of OL trnK and trnN were observed in A chunganensis andQ boulengeri These gene rearrangementsmay be explained by the theory of TDRL (Muelleramp Boore 2005 Boore 2000) In A chunganensis OL was located between trnW and trnAFor Q boulengeri the intraspecific gene rearrangements were checked in the WANCYregion Our results supported the observation that the hotspot of gene rearrangement wasadjacent to the origin of light-strand replication (San Mauro et al 2006) The widespreadoccurrence of gene rearrangement among different vertebrate groups has been examined inrelation to variability in the thermodynamic stability of the light-strand replication origin(Fonseca amp Harris 2008)

Mitochondrial metagenomic skimming by NGS could be a useful approach to recoverintraspecific rearrangements ofmitogenomes By sequencing the hotspot of gene rearrange-ment of mtDNA for Q boulengeri (from nad2 to cox1 which includes the WANCY region)four kinds of gene rearrangements were identified in spiny-bellied frog populations (Xiaet al 2016) Yet mitochondrial genome analysis may provide more valuable evidences forinterpreting the intraspecific gene rearrangements Intraspecific rearranged variations werenot common by comparison of all mtDNA records of amphibians and reptiles in GenBank

Phylogenetic reconstruction of mitogenomic dataIn this study both the phylogenetic relationships andmitogenome organization suggest thatA wuyiensis and A ricketti were significantly distinguished from other Amolops species (Feiet al 2009) Within Amolops clade the highly supported sister relationship of A wuyiensisand A richetti was consistent with the results of Cai et al (2007) Within Quasipaa cladethe phylogenetic inferences based on mtDNA sequences revealed that all individuals ofQ boulengeri formed a monophyly with high supports sister toQ verruspinosa This resultis congruent with Che et al (2009) but different from the result of Qing et al (2012) Thephylogenetic reconstruction using whole mitogenome provides more credible result thansingle gene

Yuan et al (2016) PeerJ DOI 107717peerj2786 1018

As previous demonstration (Yan et al 2013 Qing et al 2012 Cai et al 2007 Frostet al 2006) the mitogenomic approach proved useful tools for inferring phylogeneticrelationships within Neobatrachia In the present study all clades were well resolved witha few exceptions where Bayesian posterior probabilities were no less than 090 Despitetheir fast evolutionary rate mitochondrial genomes contained a phylogenetic signal thatcould be efficiently recovered with reasonable taxon sampling (Rubinstein et al 2013)

ACKNOWLEDGEMENTSWe thank the two reviewers and the editors for their insightful suggestions and commentson the paper We thank Wei Luo Ting Liu and Xiuyun Yuan for their assistance in thelaboratory work We gratefully acknowledge technical support from the laboratory ofXiaomao Zeng

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was funded by National Natural Science Foundation of China (NSFCndash31272282 31372181 31401960 31572243) and theChina Postdoctoral Science Foundation(2014M552386) and West Light Foundation of the Chinese Academy of Sciences Thefunders had no role in study design data collection and analysis decision to publish orpreparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsNational Natural Science Foundation of China NSFCndash31272282 31372181 3140196031572243China Postdoctoral Science Foundation 2014M552386West Light Foundation of the Chinese Academy of Sciences

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Siqi Yuan performed the experiments analyzed the data contributed reagentsmateri-alsanalysis tools wrote the paper prepared figures andor tables reviewed drafts of thepaper collect the specimen in the fieldbull Yun Xia conceived and designed the experiments analyzed the data contributedreagentsmaterialsanalysis tools wrote the paper prepared figures andor tablesreviewed drafts of the paper collect the specimen in the fieldbull Yuchi Zheng conceived and designed the experiments wrote the paper reviewed draftsof the paperbull Xiaomao Zeng conceived and designed the experiments contributed reagentsmaterial-sanalysis tools wrote the paper reviewed drafts of the paper

Yuan et al (2016) PeerJ DOI 107717peerj2786 1118

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

All work with animals was conducted according to relevant national and internationalguide-lines All animal care and experimental procedures were approved by the AnimalCare andUse Committee (Permit Number CIB-20121220A) Chengdu Institute of BiologyChinese Academy of Sciences

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

All field work with animals was conducted according to relevant national andinternational guide-lines Chengdu Institute of Biology issued permit number CIB2014-36 and CIB2014-110 for field work

DNA DepositionThe following information was supplied regarding the deposition of DNA sequences

The sequences of Quasipaa boulengeri and Amolops chunganensis are accessible viaGenBank accession numbers KX645665ndashKX645666 in this study respectively

Data AvailabilityThe following information was supplied regarding data availability

Sequence Read Archive (SRA httpwwwncbinlmnihgovTracessra)SRR3929655 for Quasipaa boulengeriSRR3929656 for Amolops chunganensis

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj2786supplemental-information

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HuangM Lv T Duan R Zhang S Li H 2016 The complete mitochondrial genome ofRhacophorus dennysi (Anura Rhacophoridae) and phylogenetic analysisMitochon-drial DNA Part A 27(5)3719ndash3720 DOI 1031091940173620151079873

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