2 phyla not aligning

mothur bugs
1

Hi MOTHUR team,

2 phyla do not seem to align well, specifically Chloroflexi (Thermomicrobium roseum) and Fibrobacter (F.succinogenes), while using the silva.bacteria.fasta as template. The aligning procedure picks incorrect templates to align against. However, the green genes alignment template does pick the correct template for these two phyla.

Thanks very much,
Ameet

2

If you can get me the accession numbers for better Chloroflexi and Fibrobacter sequences we can supplement the current set in the silva.bacteria.fasta alignment.

3

The accession numbers of sequences in my synthetic community mix that do not align are:

  1. Thermomicrobium Roseum (M34115) (Chloroflexi)
  2. Fibrobacter succinogenes S85 (M62696)

Both sequences are from completed genomes.

Thanks very much for your help.
Ameet

4

To follow up on the other thread - I’d suggest going into SILVA and pulling down these sequences and concatenating them to the end of the silva.bacteria.fasta file and re-doing the alignment.

5

Thanks very much Pat.

Another question: One of the user forum threads was about archaea template for chimera check. I am trying to figure out how to do this using the archaeal sequences in the SILVA NR database.

From what I understand the sequences need to be 1% apart. Are there any other guidelines? Is there a paper where there is information on what other criteria went into creating silva.gold.align? Could you please point me to that paper.

Thanks very much for your help.

6

http://genome.cshlp.org/content/21/3/494.long

7

I am using the silva.archaea.fasta to align my 454-sequences. I ran: mothur > align.seqs(reference=silva.archaea.fasta, processors=2)
and I got this message: Some of you sequences generated alignments that eliminated too many bases, a list is provided in CronBArch.trim.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well. How do I flip the parameter? Or, do I need to re-orientate my sequences?

Brandi Cron

8

I am using the silva.archaea.fasta to align my 454-sequences. I ran: mothur > align.seqs(reference=silva.archaea.fasta, processors=2)
and I got this message: Some of you sequences generated alignments that eliminated too many bases, a list is provided in CronBArch.trim.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well. How do I flip the parameter? Or, do I need to re-orientate my sequences?

Brandi Cron

9
  1. align.seqs(reference=silva.archaea.fasta, processors=2, flip=T)

or…

  1. reverse.seqs(fasta=whatever.fasta)

or

  1. trim.seqs(, flip=T)

The first approach will try the reverse complement if it looks like individual sequences are backwards, the second will get you the reverse complement for all of the sequences, and the third will get you the reverse complement when you are removing barcodes. In general, if you are using directional primers (e.g. with 454) then you should know which direction your sequences are in and should be able to do everything with option 3 above.