reverse_primer truncate and remove

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Koirobi Haldar

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Jul 14, 2013, 1:48:05 PM7/14/13
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Hello
I have 16S 454sequences which are sequenced unidirectionaly from the forward primer. I want to remove the reverse primer from my sequences. Do I put the reverse complement sequence of reverse primer or the reverse primer sequence in my mapping file?(The primer sequence and the raw read sequence with the primer sequence highlighted is given below).
 
If I use the reverse primer "Truncate_remove" option in the split_library process and use this  for denoising process. will it be alright?

my primer sequence are

Forward primer- 5' GCCAGCAGCCGCGGTAA 3'

Reverse primer- 5' CTTGTGCGGGYCCCCGTCAAT 3' Reverse complement- 5' ATTGACGGGGYCCCGCACAAG 3'
My raw sequences look

>H7K8FOK01APOTI length=433 xy=0175_1876 region=1 run=R_2013_04_10_07_29_38_

ACGCTCGACAGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTATCCGGATTTATTG

GGCGTAAAGCGAGCGCAGGCGGTTAGATAAGTCTGAAGTTAAAGGCTGTGGCTTAACCAT

AGTACGCTTTGGAAACTGTTTAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTA

GCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGT

AACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA

CGCCGTAAACGATGAGTGCTAGGTGTTGGGTCCTTTCCGGGACTCAGTGCCGCAGCTAAC

 

GCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGG

GGACCCGCACAAG
 
 

Tony Walters

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Jul 14, 2013, 1:59:27 PM7/14/13
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Hello Koirobi,

This version of the primer is the one you want to use in the ReversePrimer data fields: CTTGTGCGGGYCCCCGTCAAT

If you want to denoise and remove the reverse primer, it's best to skip reverse primer removal during split_libraries.py, do the denoising, and then run truncate_reverse_primer.py on the denoised sequences (the reason for this is the denoiser only uses the labels of the sequences generated from split_libraries.py to determine which ones were filtered out due to other quality issues, the sequences themselves (including the reverse primer) are rebuilt from the flowgram sff files, so you'll have the reverse primer present in the output of denoising).

-Tony


 
 

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