TRANSCRIPTOME ANALYSIS OF AQUACULTURE SPECIES
Transcriptome refers to the complete composition of RNAs of an
organism.
Years ago, EST analysis was the major approach for transcriptome sequencing.
Recently, RNA-seq using next-generation sequencing has allowed the most rapid
progress.
6.1 Development of EST resources of aquaculture species
1. Expressed sequence tags are single-pass sequences of
random complementary DNA (cDNA) clones from cDNA
libraries.
2. They are traditionally generated using Sanger sequencing
and therefore the resultant sequences are approximately 500
to 800 base pairs in length.
3. Several years ago, because sequencing was relatively cheap,
large numbers of ESTs can now be generated at a reasonably
low cost from either the 5′ or 3′ end of a cDNA clone to get an
insight into transcriptionally active regions.
4. ESTs were used as a primary resource for human gene
discovery (Adams et al., 1991). Thereafter, there has been an
exponential growth in the generation and accumulation of EST data
in public databases for various organisms, with approximately 74
million ESTs now available in these databases
([Link] March 2016 release number
130101, all species).
5. EST analysis is an effective genomic approach for rapid
identification of expressed genes, and has been widely used in
genome-wide gene expression studies in various tissues,
TRANSCRIPTOME ANALYSIS OF AQUACULTURE SPECIES
developmental stages and under different environmental
conditions (Franco et al., 1995).
6. In addition, the availability of cDNA sequences has accelerated
further molecular characterization of genes of interest and
provided sequence information for microarray construction and
genome annotation (Rise et al., 2004).
7. Gene expression analysis plays an important role in identifying
differentially expressed genes under different
environmental conditions and gene expression regulation,
shedding light on gene functions.
8. EST analysis has been demonstrated effective for detection of
differential expression and regulation of certain genes. Without
normalization or subtraction in library construction, the number
of the sequenced ESTs for a given gene reflected the
abundance of the gene expression at the corresponding
scenario (e.g. environmental conditions, developmental stages,
treatments).
9. Direct EST sequencing is inefficient in discovery of rarely
expressed genes. To solve this problem, the method to
construct normalized cDNA libraries was developed (Bonaldo,
Lennon and Soares, 1996; Soares et al., 1994). The basic
principle is using hybridization to reduce redundant genes
and increase the representation of rarely expressed genes.
10. However, with the adoption of RNA-seq technology, as
described below, it is unlikely that additional EST analysis will
be conducted because of the low efficiency as compared with
RNA-seq.
TRANSCRIPTOME ANALYSIS OF AQUACULTURE SPECIES
11 .Initial annotation of ESTs can be conducted by simple sequence similarity
comparisons. Further annotation analysis can be carried out after obtaining the
consensus sequences (putative unigenes), such as determination of gene
identity based on homology search, open reading frame identification, gene
ontology annotation and gene-enrichment analysis (e.g. Nakaya et al., 2007).
In order to assign gene identity to contigs and singletons, homology search is widely used. Such
an approach is especially helpful for newly studied species. BLAST is the most widely used
program to obtain high throughput EST analysis and annotation results. The BLAST package
provides different flavours of algorithms for sequence similarity searching. BLASTX is used to
search against protein database by translated consensus EST sequences, while BLASTN is used
to search against nucleotide sequence databases. The National Center for Biotechnology
Information (NCBI), ENSEMBL and Swiss-Prot are three important databases for BLAST
search. For instance, the Swiss-Prot database has a fully manually curated and annotated unigene
database, Uniprot, which can be used for identifying putative function for unigene by BALSTX.
The NCBI provides a dbEST database that can be used to search novel transcripts by BLASTN.
The dbEST is a main EST resource database, including ESTs for over 200 aquaculture species.
The ENSEMBL database can provide chromosome location information of genes, which is a
useful tool for comparative genome analysis. However, the BLAST sequence similarity
comparison provides only sequence