454 sequencing of the rehmannia tuberous root
Sequencing and transcriptome analysis of rehmannia tuberous root
The rehmannia cDNA library was constructed using a modified SMART cDNA synthesis protocol:a modified poly(T) primer with a BsgI site(5'-AAGCAGTGGTATCAACGCAGAGTACT(20)VN-3') was used in this study to replace the normal poly(T) primer. After the double-strand cDNAs were synthesized by LD-PCR, it was treated with BsgI (NEB, MA, USA) to remove the poly(A/T) tail. It should be noted that due to unknown reason a small portion of sequences contain fragmentary SMAT primer sequences (i.e. part of the entire primer sequences) in the middle of the sequence. These sequences are most likely to be generated by PCR amplification when using SMART™ PCR cDNA Synthesis protocol. We also find that some sequences deposited in GenBank also contain such partial primer sequences. Since they exist in the middle of the sequences; anyone who wants to use these sequences should be careful when carrying out assembly job.   SMART primers that should be cleaned: SMART II™ A Oligonucleotide 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG3'; Modified Poly(T) primer: 5'-AAGCAGTGGTATCAACGCAGAGTACT(20)VN-3'
In order to discover genes involved in terpenoid biosynthesis and transcription regulation, a cDNA library generated from the tuberous roots of Rehmannia glutinosa was sequenced by the 454 GS FLX titanium platform.A half plate GS-FLX Titanium run yielded 88.4MB bases from 374,444 reads.