Molecular verification of species identity, and quality of long-PCR amplicons

A sample of total genomic DNA was isolated from a single adult male of H. contortus (McMaster strain) for the sequencing of the complete mt genome. In order to ensure the specific identity of the specimen prior to sequencing, the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA was amplified from the genomic DNA sample by the PCR and sequenced directly. The sequence obtained was identical to the ITS-2 sequence published previously for H. contortus (GenBank accession number X78803; [24]). Subsequently, the complete mt genome was amplified by long-PCR from the genomic DNA in two overlapping regions (~5 kb and ~10 kb, respectively) [14]. Each amplicon appeared as one abundant band of the appropriate size on an agarose gel. Short tags (300–400 bp) were sequenced from the 10 kb and 5 kb amplicons (within the cox1 and rrnL genes, respectively) to verify their specificity and identity. Following DNA quantitation for each amplicon, the two amplicons (5 μg from each) were pooled and sequenced directly in a single reaction using 454 technology (whole genome sequencing protocol).

Validation of sequencing via 454 technology by comparison with data mined from public databases

The recently developed 454 technology platform [18] has been utilized for the sequencing of the complete nuclear genomes from a range of organisms [22, 25–28]. Although, prior to the present study, it had not been applied to mt genomes of nematodes, a study of marine microbes [22] has shown this technology (together with the Sanger method) to be particularly suited for sequencing AT-rich regions with complex secondary structures, yielding higher quality sequences and costing less overall to carry out than Sanger sequencing alone. This information indicated that 454 technology was perfectly suited to the AT-rich mt genomes of strongylid nematodes, but this required verification.

The mt genome sequence (designated HcMG-454; accession number EU346694) of H. contortus determined using 454 technology was automatically assembled into one contig of 14,055 bp. This sequence was then compared with extensive mt gene sequences available in public databases. For this comparison, ESTs (n = 24,014) from 12 different cDNA libraries, constructed from various lifecycle stages or tissues of H. contortus [29] available via [30] (n = 10,000 ESTs) and [31] (n = 14,014 ESTs) were mined for mt gene data. In total, 257 ESTs were obtained (Table 1) and found to have high similarity (90–100% at the nucleotide level) to the sequences of protein coding genes of the H. contortus in the mitochondrial genome determined in the present study. An examination of the number of ESTs obtained for each protein coding mitochondrial gene (Figure 1) revealed that cox1, is much more highly represented than any other gene (96 ESTs). The cox3, nad5 and nad4 genes are also highly represented (41, 31 and 20 ESTs, respectively). Given the number of EST reads contained within these libraries (~24,000) and the random nature in which EST reads are generated (randomly selected cDNA clones), it seems reasonable to infer that the relative proportion of the currently available EST contigs for H. contortus which match each mitochondrial gene are related to the relative abundance of the mRNA transcripts for each gene in vitro.

Gene	Gene Length (bp)	ESTs	Read Length (bp)	Identitya
nad1	879	10	118–586	95–99%
nad2	846	10	266–437	92–99%
nad3	336	2	193–229	97–98%
nad4	1230	20	203–563	93–98%
nad4L	234	4	105–110	91–97%
nad5	1582	31	237–597	96–99%
nad6	447	11	124–347	90–100%
cox1	1576	96	166–791	94–100%
cox2	693	8	293–663	96–100%
cox3	766	41	221–604	94–97%
cytb	1113	14	157–596	94–99%
atp6	600	10	266–429	92–97%

^aBased on unannotated (raw) EST data from public databases

In addition to mining EST data from online databases, GSS data (available via [30]) were mined for mt gene sequences using an unpublished cox1 sequence as an in silico-bait. We discovered contigs 002363 (14,442 bp) and 002480 (14,814) which were then mapped against HcMG-454 at both the nucleotide and amino acid levels (for protein coding genes), achieving complete coverage with > 99.7% similarity at the nucleotide level. Of the nucleotide differences, 6 were found within protein coding genes. Four of these single nucleotide differences between HcMG-454 and contigs 002363 and 002480 were interpreted to represent intraspecific variation in the nucleotide sequence of the mt genome of this species, as none resulted in an amino acid change. Two appeared to be sequencing errors, as they resulted in frameshifts in the inferred amino acid sequence: both were found in the cox1 gene and were base-called using alignments against available EST data (see Materials and methods section). All other nucleotide alterations (n = 34) were within non-coding regions.

Characteristics of the mt genome of Haemonchus contortus and comparative analysis with those from related nematodes

The complete mt genome sequence of Haemonchus contortus, HcMG-454, was characterized (Figure 2). It was 14,055 bp in length and was assembled as a single contig from 5,965 overlapping sequence reads within the 454 sequencing platform. The mt genome of this species is ~300–400 bp larger than those characterised for related species (Strongylida), which range from 13.6–13.7 kb [32, 33], but is within the range of previously published mt genomes for chromadorean nematodes (~13.6–14.3 kb) [32, 34–37]. The larger size relates primarily to an apparent expansion of the AT-rich control (465 bp versus 268, 173 and 304 bp for the corresponding region in Ancylostoma duodenale, Necator americanus and Cooperia oncophora, respectively) as well as the presence of slightly longer non-coding regions between many of the transfer RNA genes. The mt genome sequence of H. contortus is AT-rich (78.1%) and exhibits an asymmetrical nucleotide usage: 33.2% for A, 44.9% for T, 15.5% for G and 6.5% for C in the coding strand (Table 2). Within the Strongylida, AT-richness in the mt genome has been found to range from 76.6–77.2% [32, 33], and, as is observed here for H. contortus, T is the most dominant nucleotide in the coding strand (47.1–49.2%) [32, 33]. Given the AT bias (~78%) in the mt genome of H. contortus, there is a considerable bias in codon usage. Consequently, ATA (Methionine/translation initiation), ATT (Isoleucine), TTA (Leucine), and TTT (Phenylalanine) were the most commonly used codons (5.6, 6.9, 8.2 and 11.0%, respectively), compared with those containing numerous G and/or C residues, such as CGC (Glycine), GCC (Glycine) and GCG (Glycine) (0.0, 0.1 and 0.5%, respectively). These findings are consistent with previously characterized nematode mt genomes [32, 34–37].

Species	A	G	C	T	A+T	Reference
Haemonchus contortus	33.2	15.5	6.5	44.9	78.1	Present study
Ancylostoma duodenale	28.3	16.7	6.6	48.4	76.7	[32]
Necator americanus	27.4	17.1	6.3	49.2	76.6	[32]
Cooperia oncophora	30.1	16.3	6.5	47.1	77.2	[33]

The gene content of the mt genome of H. contortus is consistent with that reported for other chromadorean nematodes [32, 34–37] in having 12 protein coding genes (the cytochrome c oxidase subunits 1–3 (cox1-cox3), the nicotinamide dehydrogenase subunits 1–6 (nad1-nad6 and nad4L), cytochrome b (cytb) and adenosine triphosphatase subunit 6 (atp6)), 22 transfer RNA (tRNA) genes (Figure 3) and the small (rrnS) (Figure 4) and large (rrnL) ribosomal subunits (Figure 5). As for other chromadorean nematodes studied to date [4], no adenosine triphosphatase subunit 8 (atp 8) gene is present, and all genes are predicted to be transcribed from the same strand and in the same direction. Examination of the overall genome structure revealed that the H. contortus mt genome has gene arrangement GA2 (see Figure 2 in ref. [32]) which is consistent with all previously published mt genomes for the Strongylida [32, 33] and some Rhabditida (including Steinernema carpocapsae [38] and Caenorhabditis elegans [39]).

All circular nematode mt genomes described to date have been found to contain 22 trn genes [32, 34–37], and H. contortus is no exception (Figure 3). The trn genes for this species range from 53–61 bp, which is consistent with other nematodes [32, 34–37]. The secondary structure of the trn S genes consists of a 7–8 bp amino-acyl arm and a 5 bp anticodon stem, with a T/U residue always preceding, and a purine always following the anticodon. Twenty of the 22 trn S genes were found to have a 3–4 bp DHU arm with a 5–8 bp DHU loop and a 6–11 bp TV-replacement loop instead of a TψC arm. The two exceptions to this are the two serine (AGN and UCN) trn S genes which have a 6 bp DHU replacement loop instead of the DHU arm, and a 3–4 bp TψC arm with a 4–5 bp TψC loop instead of the TV replacement loop. These findings are consistent with the trn S genes described for all 11 chromadorean nematodes characterised to date [32, 34–37].

The rrnS and rrnL genes of H. contortus were consistent in length (702 and 915 bp, respectively) with those reported previously for most nematodes [32, 34–37], showing 77–78% and 75–76% sequence similarity, respectively, with homologous sequences from An. duodenale, N. americanus and Co. oncophora. The secondary structures for rrnS (Figure 4) and rrnL (Figure 5) were inferred by mapping against the appropriate secondary structures for N. americanus [32], originally based on Escherichia coli models [40] and predicting, either manually or through computer assisted predictions (MFOLD), changes caused by various mutations.

In accordance with the two known hookworm mt genomes [32], the rrnS structure consists of four relatively conserved domains (A-D, Figure 4) bound by numerous conserved helices [40]. A 7 nt insert between positions 187–195, apparently interacts with a 7 nt tract between positions 305–311 (see Figure 4) and is predicted to result in a slight alteration of the secondary structure between conserved elements 20 and 25 [40], resulting in an extension of the stem between elements 20 and 22 and the formation of a 4 bp and a 7 bp stem loop between elements 23 and 25 (see Figure 4). Although the secondary structure of the rrnS of Co. oncophora was not characterised previously [33], the sequence did not reveal the 7 nt tract identified in H. contortus. Whether this inferred alteration in the rrnS structure in H. contortus is unique to this and/or to closely related species remains to be elucidated.

The rrnL secondary structure predicted for H. contortus is consistent with those predicted for An. duodenale and N. americanus [32] and consists of four major stem-loop domains (1–4) (Figure 5) which appear to be conserved [32, 34–37]. The amino-acyl trn binding sites (A) and peptidyl-transferase sites (P), first described from Escherichia coli by Noller et al. [41] and later recognized in nematodes [32], are present. Hu et al. [32] found that, although these "A" and "P" binding sites were present in hookworms, the exit site (E) proposed by Noller et al. [41] was not found in the rrnL of the hookworms or Ascaris sp., Ca. elegans, Onchocerca volvulus or Trichinella spiralis [39, 42, 43]. Also, no E site was found within the secondary structure of the rrnL of H. contortus, demonstrating consistency with previous findings and lending further support to the hypothesis that many nematode rrnL genes do not have a recognizable exit site [32] or, at least, not one comparable with that of E. coli [41]. Although the secondary structure of the rrnL of Co. oncophora has not been reported previously [33], the sequence similarity (75–79%) among the rrnL genes of H. contortus, An. duodenale and N. americanus suggests a conserved secondary structure for these Strongylida.

The 36 genes of the H. contortus mt genome comprise ~94% of the entire mt genome. The remaining ~6% consists of non-coding regions, which are predicted to be functionally involved in the regulation of transcription, translation and/or replication [3, 4, 32]. The largest of these non-coding regions is the AT-rich region, which is 465 bp in length (Figure 6). As for other nematodes with the gene arrangement GA2 [3, 4, 35], this region was located between the nad5 and nad6 genes. Also, like other GA2 nematodes, the AT-rich region in H. contortus is flanked (5') by the trnA gene and (3') by the genes trnP and trnV, and a short (~35 bp) non-coding region between genes trnP and trnV. This region is 157–292 bp longer than the corresponding region reported for An. duodenale (278 bp), N. americanus (173 bp) [32] and Co. oncophora (308 bp) [33]. However, as described by Hu et al. [3], the AT-rich region is likely to exhibit significant intraspecific length polymorphism. As for hookworms [32], in H. contortus, this region exhibits a complex stem-loop formation (Figure 6), hypothesized to be involved in mt replication in the mt genome [32]. In addition to having complex secondary structure, the AT-rich region can contain repetitive elements. The AT-rich region of Ca. elegans contains six repeats (CR1-CR6) of an identical 43 nucleotide sequence [39]. Although such a repetitive element was not found in the mt genomes of An. duodenale or N. americanus [32], regions believed to represent remnants of these repeats have been found in the mt genome of Ascaris sp., in the same study as that of Ca. elegans [39]. In the present study, the AT-rich region of H. contortus does not contain the repetitive element described for Ca. elegans. However, a sequence alignment of the AT-rich regions from these two species did indicate that some of the "TAT" portions [32] of the repetitive elements described for Ca. elegans may be present. Lastly, Hu et al. [32] described a poly-A (n = 7–8) region within the AT-rich region of all chromadorean nematodes characterised at the time. A similar poly-A region is present in the AT-rich region of H. contortus (see Figure 6). It has been hypothesized [32] that such poly-A region are involved in mt gene replication, as proposed for poly-T regions found in the control region for some vertebrate and insect species [44–46]. This proposal warrants experimental investigation.

Another non-coding region between cox1 and nad4 has been described from all previously characterized nematodes exhibiting gene arrangement GA2 [32, 39], namely An. duodenale, N. americanus, As. suum, S. carpocapsae and Ca. elegans. In the latter three nematodes, this region forms a stem-loop structure [39]. However, the region does not appear to form a stem loop structure in An. duodenale nor in N. americanus [32], and does not appear to form a stem loop in the H. contortus mt genome reported herein. In addition, nematodes with gene arrangement GA3, presently represented by the filarial nematodes (B. malayi; accession no. AF538716), O. volvulus [42] and D. immitis [36]) from which complete mt genomes have been described, also have a gene order juxtaposed such that the cox1 gene follows the 3' end of nad4. There is no non-coding region between these two genes. This information suggests that if secondary structure in the non-coding region between cox1 and nad4 is involved in regulating replication, transcription and/or translation, it is not a universal requirement for all nematodes. Hu et al. [32] found a third non-coding region between the genes nad3 and nad5 (80 bp and 55 bp for An. duodenale and Ne. americanus, respectively). A similar region is present in H. contortus. In An. duodenale, this region was inferred to form a 6 bp and a 9 bp stem-loop [32], and it is proposed to be involved in interactions with RNA processing enzymes, as has been reported for similar stem-loop structures in humans [47]. No such secondary structure was found for Ne. americanus [32], nor was it predicted here for the corresponding region in H. contortus. The degree to which such structures are involved in regulating molecular events in the mitochondria of nematodes in not yet clear and is an exciting area for future research.

454 technology as a high-throughput method for sequencing mitochondrial genomes

The present study demonstrates the utility of the 454 technology platform for the sequencing of AT-rich mt genomes combined with mapping against a scaffold of available sequences mined from public EST and/or GSS databases. This approach represents a "scaled-down" version used for sequencing complete nuclear genomes [16, 18, 20, 22, 48] and overcomes the significant limitations of sequencing AT-rich templates using conventional approaches [14]. Presently, the equipment costs associated with this platform are likely to be prohibitive for most laboratories. Consequently, most laboratories will likely utilise commercial services if employing this method. In terms of cost, sequencing of a small number of mt genomes using this platform is probably not directly competitive with conventional Sanger sequencing via primer walking and/or cloning. However, if one considers the vast improvements, in terms of efficiency, in relation to the complete sequencing and sequence assembly of these genomes, we contend that this approach is a practical alternative.

The period from genomic DNA extraction to the final output of a complete, assembled mt genome takes ~2–3 weeks. In research applications, where a panel of conserved primers is available for primer walking, this level of output is probably achievable. However, for applications where such primers are not available (because of a lack or absence of sequence data from the organism or a related species) and must be designed de novo based on sequencing results, this present approach has major advantages. Equally, shotgun cloning-based sequencing would not be as efficient, unless a high-throughput (e.g., robotic) system were available for the rapid isolation of large numbers of clones for plasmid purification and subsequent sequencing to ensure adequate coverage of the mt genome, thus substantially increasing costs. Furthermore, by either a primer walking or shotgun cloning approach, a significant amount of bioinformatic processing is required for contig assembly following sequencing, which is not required using the 454 platform because contig assembly is automated. Economically, if 454 technology is applied as a high-throughput system in which multiple mt genomes are sequenced simultaneously, the direct costs per such genome (~USD 1,250) becomes directly comparable with, if not less expensive than other approaches. The benefits in terms of efficiency presented by this technology are considered substantial.

In addition to cost and efficiency benefits, the 454 sequencing method may provide greater reliability in the sequence output (estimated sequencing error in the present study was 2 errors in 14,055 bp of sequence) which will likely improve as newer versions (e.g., the GS-FLX system) of the sequencing platform and assembly software are made available. Given the cost and laborious nature of primer walking and/or conventional cloning-based methods, most mt genomes presently available have been assembled as a single contig following uni- or bi-directional sequencing, resulting in a one- to two-fold coverage of the genome (forward and reverse strands). The sequencing of large numbers of overlapping sequences generated from a template using the 454 technology offers a substantial increase in coverage. In the present study, the complete mt genome was assembled from ~6,000 overlapping sequences, each read being ~100 bp [18]. This translates into a total sequence output from one reaction of ~600,000 bp, resulting in substantial "coverage" of the mitochondrial genome, which would be impractical, too laborious and costly to achieve using conventional sequencing approaches.

Previously, it has been suggested that the 454 technology may be less reliable for sequencing homopolymeric and repetitive elements [18, 22], which have been detected previously in the mt genomes of nematodes, particularly in the AT-rich region [38]. In the mt genome of H. contortus determined, 66 such regions (of 7–9 As or Ts) were identified, but there was no evidence (based on comparison with EST and GSS datasets) of any problems herein with sequencing through such elements. In the present study, the discovery of a complete mt genome sequence for H. contortus in a GSS database allowed the direct evaluation of the 454 sequencing output, and did not reveal any sequencing errors. As homopolymeric and repetitive elements can occur in the AT-rich region and elsewhere, it may be warranted, for organisms for which no prior sequence data are available, to undertake conventional Sanger sequencing to independently verify the accuracy of such sequence elements determined by 454 technology.

Production of Haemonchus contortus, isolation of genomic DNA and verification of specific identity by molecular means

Adults of H. contortus (McMaster strain) were produced in a helminth-free sheep [49]. Adult worms were isolated from the abomasum (= stomach) and washed extensively in physiological saline (25°C). Using a dissecting microscope (5× magnification), the sex of individual worms was verified microscopically, and male and female worms separated. Individual worms were transferred to sterile, screw-top cryogenic tubes (Nunc) and frozen (-70°C) in a minimal amount of buffer. After thawing, total genomic DNA was isolated from an individual male of H. contortus using a standard sodium dodecyl-sulphate/proteinase K treatment [50], followed by purification over a mini-column (Wizard, Promega). The specific identity of the specimen was verified by PCR-based amplification of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA using an established method, followed by mini-column purification of the amplicon and subsequent automated sequencing (BigDye chemistry v3.1) [51]; the ITS-2 sequence determined was identical to that with GenBank accession no. X78803 [52].

Long-PCR amplification of two mt genome regions

The complete mt genome was amplified from ~10% (20–40 ng) of the genomic DNA from the individual specimen by long-PCR (BD Advantage 2; BD Biosciences) as two overlapping amplicons (large and small), using the protocol described by Hu et al. [14] with minor modifications. The large (~10 kb) amplicon was produced using the primers MH43F (forward: 5'-TTCTTATGAGATTGCTTTTTCT-3') and MH40R (reverse: 5'-GAATTAAACTAATATCACGT-3'). Briefly, the PCR (50 μl) was conducted using 10 pmol of each of the two oligonucleotide primers, 100 μM of each dNTP, 3 mM MgCl₂ and 1 U of BD Advantage 2 Taq polymerase (BD Biosciences) using an ABI 2720 thermal cycler (ABI), employing the following cycling protocol: one cycle at 94°C for 2 min (initial denaturation), followed by 35 cycles of 94°C for 30 s (denaturation), 50°C for 30 s (annealing) and 65°C for 10 min (extension), followed by a final extension at 65°C for 10 min (utilizing the appropriate positive and negative controls). The small (~5 kb) amplicon was generated using the primer set MH39F (forward: 5'-TAAATGGCAGTCTTAGCGTGA-3') and MH38R (reverse: 5'-TAAATGGCAGTCTTAGCGTGA-3') under the same conditions, with the exception that the extension temperature was reduced to 60°C [14]. The specificity of the two primer sets had been validated previously [14]. Following the PCR, each amplicon was subjected to electrophoresis in 1% agarose, using a 1 kb DNA Ladder (Promega) to estimate size. Amplicons were then purified over a mini-column (Wizard, Promega) and quantified spectrophotometrically using a NanoDrop ND-1000 UV-VIS spectrophotometer v.3.2.1 (NanoDrop Technologies). The specificity of the PCR conditions and amplicons was verified by partial, automated Sanger sequencing (employing BigDye Chemistry v3.1), using primers COIF (5'-TTTTTTGGGCATCCTGAGGTTTAT-3') and MH28R (5'-CTAACTACATAATAAGTATCATG-3') (large fragment) and MH37F (5'-GGAGTAAAGTTGTATTTAAAC-3') and MH40R (5'-GAATTAAACTAATATCACGT-3') (small fragment) [3, 35].

Automated sequencing using 454 technology

The two amplicons (~5 kb and 10 kb; 5 μg of each) spanning the mt genome of H. contortus were pooled and subsequently sequenced using the Genome Sequencer 20 (GS 20; Roche) according to the protocol provided [18]. The mt genome sequence (designated HcMG-454; GenBank accession no. EU346694) was assembled automatically and compared against EST and GSS sequences for H. contortus available from public databases [30, 31]. HcMG-454 was scanned for open reading frames (ORFs) using ORFinder [53], employing the "Invertebrate Mitochondrial" option. Protein coding genes were identified by BLASTx analysis of the inferred amino acid sequences, and the initiation and termination codons identified by alignment at the nucleotide (ClustalX) and amino acid (Clustal W) levels against the mostly closely related nematode species for which the mt genome has been characterized. The positions and secondary structures of all transfer RNA (trn) genes were identified or determined using tRNAscan SE 1.21 [54] using the "Nematode Mito" source option and the "Invertebrate Mito" tRNA isotype prediction option. The rrnL and rrnS genes and AT-rich control region were identified by BLASTn analysis and comparisons with respective sequences within the mt genomes of An. duodenale, Ne. americanus, Co. oncophora and Ce. elegans (see GenBank accession numbers AJ417718, AJ417719, AY265417 and X54252, respectively). The secondary structures for the rrnL and rrnS were predicted by sequence alignment against rrnL and rrnS from Ne. americanus [32] with secondary structures for variable regions determined using MFOLD [55]. The secondary structure of the AT-rich region was also determined using MFOLD. Following genome annotation, each protein-coding gene was conceptually translated using Translation Tool (v.3.1) [56] using the Invertebrate Mitochondrial Code" option. Amino acid sequences were aligned with those inferred from previously characterised mt genomes for species of Strongylida (i.e., An. duodenale, Ne. americanus and Co. oncophora) and C. elegans using Clustal W [57], adjusted manually and verified using BioEdit v.7.0.3 [58].