Silva Bacteria Aligning Position

Theory behind mothur
1

Hi all,

I am a honours student working on the MiSeq SOP while waiting for the sequencing results to come back.

I would like to ask at the step of aligning the DNA sequence to the reference alignment (pcr.seq) step, using the silva bacteria database as reference, how do you know the starting position is 11894 and the ending position is 25319? Is there any command to know where the starting and ending positions are?

Moreover I am using the primer 27F-519R in the MiSeq sequencing, will there be any difference? Also, is there any other referencing database out there?

Thank you and cheers,

Alan

2

Here’s what you do…

  1. Take ecoli’s 16s and trim it to the region being amplified by your primers
  2. Align the trimmed sequence to silva.bacteria.fasta
  3. Run the *align file through summary.seqs - take note of the start and end positions
3

Hi,

Using the steps above I get this after running summary.seqs(fasta=silva.bacteria.pcr.fasta)


Start End NBases Ambigs Polymer NumSeqs Minimum: 13861 23444 230 0 3 1 2.5%-tile: 13862 23444 252 0 4 341 25%-tile: 13862 23444 253 0 4 3404 Median: 13862 23444 253 0 4 6808 75%-tile: 13862 23444 253 0 5 10212 97.5%-tile: 13862 23444 254 1 6 13275 Maximum: 13862 23491 311 5 9 13615 Mean: 13862 23444 252.963 0.0475946 4.5798 # of Seqs: 13615

I am not sure if I should consider 13861 and 23491 as my start and end positions or I should use 13862 and 23444, since the nnumber of bases obtained with the 23491 is higher than 275.

Thanks!

4

I’d probably just use 13862 and 23444
Pat

5

Thanks for the reply!
I just used the command

mothur > align.seqs(fasta=Pustular.good.unique.fasta, reference=silva.bacteria.fasta)
mothur > summary.seqs(fasta=current, count=current)

Then the summary table just listed out the start position at 1044, which is the usual start position of primer 27F-591R.

Thank you again!