rosenbergii, and the large number of genetic markers predicted should constitute an invaluable resource for future genetic research studies on M. The large transcript set assembled here represents the most comprehensive set of transcriptomic resources ever developed for reproduction traits in M. Gene expression analysis was also used to compare expression patterns from ovary and testis tissue libraries to identify genes with potential roles in reproduction and sex differentiation. After optimisation, de novo assembly resulted in 44,407 contigs with a mean length of 437 bp, and the assembled transcripts were further functionally annotated to detect single nucleotide polymorphisms and simple sequence repeat motifs. The influence of multiple k-mers on assembly performance was assessed to gain insight into transcriptome assembly from short reads. Bioinformatic analyses based on three state-of-the-art assemblers, the CLC Genomic Workbench, Trans-ABySS, and Trinity, that use single and multiple k-mer methods respectively, were used to analyse the data. A total of 13,733,210 combined raw reads (1720 Mbp) were obtained from Ion-Torrent PGM and 454 FLX. rosenbergii applying RNA-Seq technologies directed at identifying genes that have potential functional roles in reproductive-related traits. Here, we present the first hybrid transcriptome from M. rosenbergii sex-differentiation system more widely in freshwater prawns. Despite its economic importance, there is currently a lack of genomic resources available for this species, and this has limited exploration of the molecular mechanisms that control the M. Nucleotide sequence accession numbers.The giant freshwater prawn, Macrobrachium rosenbergii, a sexually dimorphic decapod crustacean is currently the world’s most economically important cultured freshwater crustacean species. The total number of predicted protein-coding genes in the CS3005 genome is 13,355. A total of 1,179 CS3005 genes from the combined Augustus and Fgenesh predictions did not have reciprocal best BLAST hits to Ph1 and were given locus tags with numbers starting from 30001 in CS3005. For example, TRI5 has the locus tags FGSG_03537 and FG05_03537 in Ph1 and CS3005, respectively. Unique gene identifiers from 12,176 Ph1 genes were transferred to CS3005 to indicate orthologous genes. BLAST reciprocal best hit analyses with ≥80% identity were performed to identify CS3005 homologues of the Ph1 gene sets (downloaded from the Broad website). The Augustus and Fgenesh predictions were then combined. To supplement the Augustus gene predictions, the masked genome sequence was then used as input into Fgenesh run in the MolQuest 2.4.3 package. Regions in which Augustus predicted genes were masked using the maskFastaFromBed script in BEDTools version 2.14.3-1 ( 13). To annotate the protein-coding genes of CS3005, Augustus version 3.0.1 ( 11) was used to ab initio predict genes with guidance from the Ph1 coding sequences provided to Augustus, following BLAT (version 35x1) ( 12) alignment (85% identity cutoff, with only unique hits retained) to the CS3005 contigs. The assembly L 50 (minimum number of contigs for which 50% of the assembly is contained) is 27 contigs, with an N 50 length of 460 kbp. A total of 36.6 Mbp of sequence was assembled into 424 contigs at an average sequence depth of 40-fold. Genomic reads were de novo assembled in CLC bio Genomics Workbench using default parameters, with the scaffolding option selected. These analyses indicated a de novo assembly, and ab initio gene predictions were required for the CS3005 genome. The assembly of the remaining genomic reads to the Ph1 reference genome suggested that only 97% of the Ph1 genome was shared with CS3005 and approximately 1 Mb of sequence was unique to each isolate. Mitochondrial reads were removed by mapping all reads to the Ph1 mitochondrial genome downloaded from the Broad Institute's Fusarium database ( ). The reads were imported into the CLC bio Genomics Workbench version 6.5.1 and quality trimmed (quality limit, 0.05, with no more than two ambiguous residues and two 5′ nucleotides removed). A total of 2.12 Gbp of raw data were generated from this sequence run. An indexed Illumina TruSeq library was prepared by the Australian Genome Research Facility, Melbourne, Australia, and sequenced using 100-bp paired-end reads on an Illumina HiSeq 2000 instrument using approximately 1/12 of a sequencing lane. Fungal DNA for sequencing was extracted from freeze-dried mycelia using a QIAgen DNeasy plant DNA minikit.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |