Open AccessOptimizing of cDNA preparation for next generation sequencing
Author(s) -
Qiaoping Qin,
Lanlan Zhang,
Nanyi Li,
Yongyi Cui,
Kai Xu
Publication year - 2011
Publication title -
yichuan
Language(s) - English
Resource type - Journals
ISSN - 0253-9772
DOI - 10.3724/sp.j.1005.2010.00974
Subject(s) - complementary dna , biology , dna sequencing , shotgun sequencing , gene , single cell sequencing , illumina dye sequencing , genetics , computational biology , cdna library , deep sequencing , genomic dna , sequence analysis , microbiology and biotechnology , genome , exome sequencing , phenotype
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.