pcr.seqs for greengenes

Commands in mothur
1

Dear all,

I am pretty new for MiSeq sequencing and 1st time to process the data. If to align and classify the sequences using the Greengenes database,especially for the pcr.seq, which type of greengene fasta i need to use?

i used gg_13_5.fasta for the pcr.seqs. Is this correct? When performing the align.seqs, it pop up an error massage saying the template is not align. The log as below:

_mothur > pcr.seqs(fasta=gg135.fasta, oligos=pcrTest.oligos)

Using 1 processors.

Output File Names:
gg135.pcr.fasta
gg135.bad.accnos
gg135.scrap.pcr.fasta

It took 574 secs to screen 1262986 sequences.

mothur >
summary.seqs(fasta=gg135.pcr.fasta)


Output File Names: gg135.pcr.summary

It took 18 secs to summarize 1097706 sequences.

mothur >
align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=gg135.pcr.fasta)

Using 1 processors.

Reading in the gg135.pcr.fasta template sequences… [ERROR]: template is not aligned, aborting.
DONE.
It took 0 to read 0 sequences._

How to solve this problem? Thank you for the advise…

Best regards,
Marcus

2

You could… 1) align the gg database to the silva reference and then use those coordinates or 2) use the primer sequences to trim the sequences in the gg database. the risk with the second option is that the primers are unlikely to match to all of the sequences in the database.

Pat