Current Location: Protocol

Sequence Assemble

Release time:2013.01    Source:

To estimate the quality of the generated sequence traces, proofreading and contig assembly of sequencing peak diagramswere performed using CodonCode Aligner 3.7.1 (CodonCode Co., USA).Base calling was carried out using the Phred program (version no. 0.020425.c). The quality values were defined for three levels: low quality (0 to 19 QV), medium quality (20 to 30 QV) and high quality (higher than 30 QV). The sequences showing >2 bases with a quality value below 20 QV in a 20-base window were trimmed. The forward and reverse reads have a minimum length of 100 bp, a minimum average QV of 30, and the post-trim lengths should be >50% of the original read length. In addition, the assembled contig should have a minimum average QV score of 40 and >50% overlap in the alignment of the forward and reverse reads.The ITS2 region can be obtained using the HMMer annotation methodbased on the Hidden Markov model (HMM) to remove the 5.8S and28S sections at both ends of the sequences.All complete “psbA–trnH” sequences are retrieved according to GenBank annotations.Subsequences marked as ITS2 or psbA-trnHintergenic spacer were recovered after deleting sequences with ambiguous nucleotides and those shorter than 100 bp.

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