Limits...
Using 454 technology for long-PCR based sequencing of the complete mitochondrial genome from single Haemonchus contortus (Nematoda).

Jex AR, Hu M, Littlewood DT, Waeschenbach A, Gasser RB - BMC Genomics (2008)

Bottom Line: A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets.The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining.This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria, 3030, Australia. ajex@unimelb.edu.au

ABSTRACT

Background: Mitochondrial (mt) genomes represent a rich source of molecular markers for a range of applications, including population genetics, systematics, epidemiology and ecology. In the present study, we used 454 technology (or the GS20, massively parallel picolitre reactor platform) to determine the complete mt genome of Haemonchus contortus (Nematoda: Trichostrongylidae), a parasite of substantial agricultural, veterinary and economic significance. We validate this approach by comparison with mt sequences from publicly available expressed sequence tag (EST) and genomic survey sequence (GSS) data sets.

Results: The complete mt genome of Haemonchus contortus was sequenced directly from long-PCR amplified template utilizing genomic DNA (~20-40 ng) from a single adult male using 454 technology. A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets. The comparison demonstrated that the 454 technology platform is reliable for the sequencing of AT-rich mt genomes from nematodes. The mt genome sequenced for Haemonchus contortus was 14,055 bp in length and was highly AT-rich (78.1%). In accordance with other chromadorean nematodes studied to date, the mt genome of H. contortus contained 36 genes (12 protein coding, 22 tRNAs, rrnL and rrnS) and was similar in structure, size and gene arrangement to those characterized previously for members of the Strongylida.

Conclusion: The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining. This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.

Show MeSH
The inferred secondary structure of the mitochondrial large ribosomal subunit (rrnL) for Haemonchus contortus. Bonds between C:G and U:A nucleotides indicated by a straight line; bonds between U:G indicated by a closed circle and between A:G indicated by an open circle as per Hu et al [32]. Binding sites for the amino-acyl trn (A), peptidyl-transferase (P) or both (AP) as defined by Noller et al. [41] indicated in bold text according to Hu et al. [32].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2254599&req=5

Figure 5: The inferred secondary structure of the mitochondrial large ribosomal subunit (rrnL) for Haemonchus contortus. Bonds between C:G and U:A nucleotides indicated by a straight line; bonds between U:G indicated by a closed circle and between A:G indicated by an open circle as per Hu et al [32]. Binding sites for the amino-acyl trn (A), peptidyl-transferase (P) or both (AP) as defined by Noller et al. [41] indicated in bold text according to Hu et al. [32].

Mentions: 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 (atp8) 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]).


Using 454 technology for long-PCR based sequencing of the complete mitochondrial genome from single Haemonchus contortus (Nematoda).

Jex AR, Hu M, Littlewood DT, Waeschenbach A, Gasser RB - BMC Genomics (2008)

The inferred secondary structure of the mitochondrial large ribosomal subunit (rrnL) for Haemonchus contortus. Bonds between C:G and U:A nucleotides indicated by a straight line; bonds between U:G indicated by a closed circle and between A:G indicated by an open circle as per Hu et al [32]. Binding sites for the amino-acyl trn (A), peptidyl-transferase (P) or both (AP) as defined by Noller et al. [41] indicated in bold text according to Hu et al. [32].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254599&req=5

Figure 5: The inferred secondary structure of the mitochondrial large ribosomal subunit (rrnL) for Haemonchus contortus. Bonds between C:G and U:A nucleotides indicated by a straight line; bonds between U:G indicated by a closed circle and between A:G indicated by an open circle as per Hu et al [32]. Binding sites for the amino-acyl trn (A), peptidyl-transferase (P) or both (AP) as defined by Noller et al. [41] indicated in bold text according to Hu et al. [32].
Mentions: 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 (atp8) 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]).

Bottom Line: A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets.The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining.This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria, 3030, Australia. ajex@unimelb.edu.au

ABSTRACT

Background: Mitochondrial (mt) genomes represent a rich source of molecular markers for a range of applications, including population genetics, systematics, epidemiology and ecology. In the present study, we used 454 technology (or the GS20, massively parallel picolitre reactor platform) to determine the complete mt genome of Haemonchus contortus (Nematoda: Trichostrongylidae), a parasite of substantial agricultural, veterinary and economic significance. We validate this approach by comparison with mt sequences from publicly available expressed sequence tag (EST) and genomic survey sequence (GSS) data sets.

Results: The complete mt genome of Haemonchus contortus was sequenced directly from long-PCR amplified template utilizing genomic DNA (~20-40 ng) from a single adult male using 454 technology. A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets. The comparison demonstrated that the 454 technology platform is reliable for the sequencing of AT-rich mt genomes from nematodes. The mt genome sequenced for Haemonchus contortus was 14,055 bp in length and was highly AT-rich (78.1%). In accordance with other chromadorean nematodes studied to date, the mt genome of H. contortus contained 36 genes (12 protein coding, 22 tRNAs, rrnL and rrnS) and was similar in structure, size and gene arrangement to those characterized previously for members of the Strongylida.

Conclusion: The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining. This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.

Show MeSH