Detection of novel SNPs in Taiwan Country chicken EST libraries
Date Issued
2008
Date
2008
Author(s)
Wang, Yi-Hui
Abstract
Single nucleotide polymorphism (SNP) is the basic DNA variation in genome as the genetic marker for biological study. A SNP locating at coding region is called cSNP, which results in two categories: non-synonymous SNP (nsSNP) with a change in corresponding amino acid and synonymous SNP (sSNP) without change of amino acid sequence. Most of the SNPs in chicken were discovered from genome sequences that are considered more accurate than expressed sequence tags (ESTs). However, the possibility of cSNP detection might be increased by using large-scale ESTs from multiple sources and tissues. The objective of this study is to predict and validate novel SNPs from Taiwan Country chicken EST libraries. This study adopted those EST libraries that were constructed from seven different tissues (pituitary gland, ovary, liver, adipose tissue, oviduct, shell gland, and muscle) of two different lines (B－meat production and L2－egg production ) of Taiwan Country chickens. The 42,404 clones of EST libraries were sequenced. These sequences were analyzed with certain quality control criteria to exclude those sequences of low quality, intron, multiple genes, short and low complexity, thus resulted in 36,463 high quality EST sequences. There were 3,455 contigs assembled along with 11,649 singlets separated after cluster analysis. The results of cluster analysis were used for sequence identity, functional annotation and SNPs prediction. For sequence identity, 15,104 unique sequences were searched by blastn against NT and RefSeq database of National Center of Biotechnology Information (NCBI). There were 10,490 unique sequences with chicken identity, in which 8,768 unique sequences were from RefSeq database. Using FatiGOplus software for functional annotation, it obtained 3,457 unique sequences with Gene Ontology (GO) annotation. The results showed the major commonly functions in the different tissues were primary metabolic process and protein binding, and the activated gene located at cell part. Besides, there were specific major functions for different tissues. The SNPs prediction was performance by Polyphred algorithm and the filtering criteria of a SNP detected in a particular contig were at least 5 EST sequences in that contig and the SNP frequency of 0.15. Those contigs and predicted SNPs were searched against Genome and dbSNP database on NCBI website for confirming their locations. In further using SNP genotyping platform for validating candidate SNPs and analyzing of genotypes. In the results, we obtained 1,107 predicted SNPs which were from 401 contigs with a predicted SNP per 340 base pairs. The number of SNPs localized at 5’ UTR, exon, intron, 3’ UTR, pseudogene, and unknown location were 60, 704, 10, 121, 5, and 207, respectively. There were 545 synonymous and 159 non-synonymous SNPs in those 704 cSNPs. In addition, only 290 out of those 1,107 predicted SNPs were found in dbSNP database using blast. The results of SNP validation and genotyping analysis were showed the accuracy of prediction was 91% and partial genotype frequency of candidate SNPs were significant difference between two lines of Taiwan Country chickens. According to the preliminary results, the analysis pipeline and strategy which used in this study was effective for SNP prediction in Taiwan Country chickens, and facilitating to select candidate SNPs for further study. The predicted SNPs might have certain effects on the functionality of those genes in which they locate. However, it is necessary to validate these predicted SNPs by certain molecular biology methods for enhancing the accuracy and practicability in Taiwan Country chickens industry.
Subjects
Genotyping
Single nucleotide polymorphism
Bioinformatic tools
Expressed sequence tag
Taiwan Country chicken
Type
thesis
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