Verification of sequences derived from de novo assembly of mRNA Seq. data of Gracilaria changii

De novo RNA –Seq assembly facilitates the study of transcriptomes without the need for a genome sequence especially for non-model organisms. However, assembly of such data may produce errors. One of these errors could be due to direct and inverted repeats in sequences. However, the presence of repeat contents could be due to either assembly problem or post transcriptional modification in the cells. This study was conducted to verify the sequences (1_CL2250Contig1 (Gc2250), CC_1_CL1944Contig1 (Gc1944), 1_CL8489Contig1 (Gc8489) and CC _Lc_4470 (Gc4470)) derived from de novo assembly of mRNA sequencing data of Gracilaria changii which contain direct and inverted repeats. The cDNA of control and sulphate deprivated G.changii samples were amplified by polymerase chain reaction (PCR) using specific primers. The PCR products were cloned into the TOPO TA vector and transformed into Escherichia coli DH5α. Colony PCR was performed for confirmation of positive transformants. The TOPO TA vector carrying the inserted sequence was extracted from the host. For each PCR products, plasmid DNA samples from three positive clones were sequenced using M13 universal primer. The sequencing results showed that, the repeats were due to the assembly errors.

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