[Optimizing of cDNA preparation for next generation sequencing].
Next generation sequencing has already been used for genomic analysis of microorganis, human being, animals, and plants. Sample preparation is prerequisite and most important for large-scale sequencing. There are two major interferences for large-scale sequencing, polyA and abundant genes' concealment for rare genes. In order to solve these problems, we used total RNA extracted from violaceae leaves to produce double stranded cDNA. DSN nuclease was used to treat the ds cDNA prior to removing the polyA. Randomly sequencing 100 clones of the treated cDNA showed that there were 94 independent clones in the treated sample, and the sequences did not contained polyA. However, only 62 independent clones were found in the untreated sample, and 15 of the sequencing files were affected by polyA. By randomly sequencing of the treated cDNA, we also found two clones encoded two interested genes. We failed to isolate these genes although the protein mass peaks of them had been found in the MALDI-TOF trace. Furthermore, we designed primers from two known genes with different expression abundances. The PCR yields were approaching similar using the treated cDNAs as templates. These results showed that, removal of the polyA and enrichment of rare genes with DSN can meet the requirements of large-scale sequencing and discovery of new genes.
Qin
Qiao-Ping
QP
School of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Lin'an 311300, China. qinqp@zafu.edu.cn
Zhang
Lan-Lan
LL
Li
Nan-Yi
NY
Cui
Yong-Yi
YY
Xu
Kai
K
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis* / Q000096:biosynthesis; D015723:Gene Library; D005796:Genes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods / Q000639:trends*
D017931:DNA Primers; D018076:DNA, Complementary