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Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 pyrosequencing.

Iranawati F, Jung H, Chand V, Hurwood DA, Mather PB - Int J Mol Sci (2012)

Bottom Line: Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences.One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively.Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

View Article: PubMed Central - PubMed

Affiliation: Earth, Environmental and Biological Sciences, Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland 4000, Australia; E-Mails: v.chand@qut.edu.au (V.C.); d.hurwood@qut.edu.au (D.A.H.); p.mather@qut.edu.au (P.B.M.).

ABSTRACT
Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

No MeSH data available.


Related in: MedlinePlus

Summary of H. siamensis 454 pyrosequencing sequences.
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f1-ijms-13-10807: Summary of H. siamensis 454 pyrosequencing sequences.

Mentions: Roche 454 GS-FLX sequencing and contig assembly of GSSs prepared from H. siamensis gDNA that was purified from fin tissue were undertaken. Sequences that passed basic quality standards were clustered and assembled de novo. The 454 sequencing run produced a total of 65,954 GSS sequences (total = 17.44 Mb) from gDNA isolated from pooled fin tissue samples. Average GSS length was 264 nucleotides (nt). Assembly of high quality GSSs generated 857 contigs averaging 352 nt in length from 5297 GSSs (Table 1). Most GSSs were unassembled as singletons comprising a total of 46,393 ESTs (total = 12.07 Mb) with an average GSS length of 274 nt. Total average GSS read length (264 nt) were longer than previous studies have reported in similar 454 sequencing runs for EST in non-model species including for Glanville fritillary (197 nt [19]), cichlid fishes (202 and 206 nt [25]) and European hake (206 nt [28]), but lower than for channel catfish (292 nt [29]), giant freshwater prawn (311 nt [21]) and bream (367 nt [14]). Different average read lengths may result from the total number of raw reads and different target material (mRNA vs. gDNA) subjected to sequencing. As shown in Figure 1, assembly of high quality H. siamensis GSS sequences generated 857 contigs varying in length from 101 nt to 2373 nt (average 352 nt; total 301,534 nt), with 143 (17%) being >500 nt in length. Singletons ranged from 50 nt to 742 nt in length (average 274 nt, total 12,706,536 nt) (Figure 1). To our knowledge, this is the first large scale study of genomic data from H. siamensis.


Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 pyrosequencing.

Iranawati F, Jung H, Chand V, Hurwood DA, Mather PB - Int J Mol Sci (2012)

Summary of H. siamensis 454 pyrosequencing sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472715&req=5

f1-ijms-13-10807: Summary of H. siamensis 454 pyrosequencing sequences.
Mentions: Roche 454 GS-FLX sequencing and contig assembly of GSSs prepared from H. siamensis gDNA that was purified from fin tissue were undertaken. Sequences that passed basic quality standards were clustered and assembled de novo. The 454 sequencing run produced a total of 65,954 GSS sequences (total = 17.44 Mb) from gDNA isolated from pooled fin tissue samples. Average GSS length was 264 nucleotides (nt). Assembly of high quality GSSs generated 857 contigs averaging 352 nt in length from 5297 GSSs (Table 1). Most GSSs were unassembled as singletons comprising a total of 46,393 ESTs (total = 12.07 Mb) with an average GSS length of 274 nt. Total average GSS read length (264 nt) were longer than previous studies have reported in similar 454 sequencing runs for EST in non-model species including for Glanville fritillary (197 nt [19]), cichlid fishes (202 and 206 nt [25]) and European hake (206 nt [28]), but lower than for channel catfish (292 nt [29]), giant freshwater prawn (311 nt [21]) and bream (367 nt [14]). Different average read lengths may result from the total number of raw reads and different target material (mRNA vs. gDNA) subjected to sequencing. As shown in Figure 1, assembly of high quality H. siamensis GSS sequences generated 857 contigs varying in length from 101 nt to 2373 nt (average 352 nt; total 301,534 nt), with 143 (17%) being >500 nt in length. Singletons ranged from 50 nt to 742 nt in length (average 274 nt, total 12,706,536 nt) (Figure 1). To our knowledge, this is the first large scale study of genomic data from H. siamensis.

Bottom Line: Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences.One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively.Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

View Article: PubMed Central - PubMed

Affiliation: Earth, Environmental and Biological Sciences, Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland 4000, Australia; E-Mails: v.chand@qut.edu.au (V.C.); d.hurwood@qut.edu.au (D.A.H.); p.mather@qut.edu.au (P.B.M.).

ABSTRACT
Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

No MeSH data available.


Related in: MedlinePlus