I used primer 515F and 806R to select the v4 region from the E coli 16S rRNA sequence list on the mothur SOP, and align the v4 region to silva.seed_v132.align. However, the summary showed that the start and end position are both -1. Does anyone know what could be wrong here?
Hmmm, I’m not sure what you’re doing to generate the custom alignment. Can you provide more detail? For what it’s worth, the numbers in the MiSeq SOP are for the V4 region. You should be able to use those without a problem.
I was just trying to see if I can customize the Silva seed reference by myself, because I might need to use reference alignment for other regions.
I used primer 515F GTGYCAGCMGCCGCGGTAA and 806R GGACTACNVGGGTWTCTAAT to trim the E coli 16S rRNA sequence provided on the Miseq SOP, and I aligned this V4 sequence against silva.seed_v132.align, and here is the summary report. Thanks for your kind help!
Reading in the /home/zhinew//UMich whole genome sequencing project/SILVA database/silva.seed_v132.align template sequences… DONE.
It took 7 to read 11180 sequences.
Aligning sequences from /home/zhinew//UMich whole genome sequencing project/SILVA database/Ecoli.v4.fasta …
1
It took 0 secs to align 1 sequences.