How can I extract Archaeal sequences also ?

Hello Mothur members.

I have amplified the V4-V5 hypervariable region of 16 S rRNA gene using the primer sets specific to both Bacteria and Archaea. I follow MiSeq SOP where SILVA-based bacterial reference alignment is provided. I want to also extract the archaeal ssequences from my samples. Could you please suggest how I can do so ?
(My OS is Windows 64 BIT, and mothur version is 1.33.3)

Looking forward,

After you’ve classified your sequences you can extract particular lineages using the get.lineage() command.

For example,

classify.seqs(fasta=yourfile.fasta, count=yourfile.count_table, taxonomy=trainset9_032012.pds.tax, template=trainset9_032012.pds.fasta)
get.lineage(fasta=yourfile.fasta, count=yourfile.count_table, taxonomy=yourfile.pds.wang.taxonomy, taxon=Archaea)

This works with any of the classification databases, you just need to modify your name accordingly, for example in Greengenes it’s ‘k__Archaea’.

Thank you very much for the explanation.

In case if I want to allign and classify bacteria and Archaea together (in one step in mothur), what shoud I do? This is because I want to see archaeal and bacterial otus combined in one picture.

Which reference file should I use for doing this, so that I can allign bacteria and archaea together?

Looking forward

You need to use something like greengenes or silva where both archaea and bacterial sequences are included.

Hello Dr. Schloss,

Thanks for your suggestion.

  1. In the silva and greengenes reference files posted in http://www.mothur.org/wiki/Silva_reference_files and http://www.mothur.org/wiki/Greengenes-formatted_databases respectively, where can I find fasta file to be fed at PCR.seq (or allign.seq) and for classify.seqs command ? I am sorry but I got confused while switching reference files provided in Miseq SOP to these reference files.

  2. I read in http://www.mothur.org/wiki/Greengenes-formatted_databases ,about greengenes reference allignment, that it should be used (if necessary, but why!?) to align sequences. You also suggest not to use it for real analysis due to poor allignment. So, what can be used for allignment in place of this if one is using greengenes.

  3. My second question is while using greengenes, how can I find start and end position of my sequences that has to be fed at pcr.seqs command line ?

Looking forward,

Richa




looking forward,

Richa

  1. In the silva and greengenes reference files posted in > http://www.mothur.org/wiki/Silva_reference_files > and > http://www.mothur.org/wiki/Greengenes-f > … _databases respectively, where can I find fasta file to be fed at PCR.seq (or allign.seq) and for classify.seqs command ? I am sorry but I got confused while switching reference files provided in Miseq SOP to these reference files.

If you download the compressed files from there you’ll find fasta formatted align files that you can use.

  1. I read in > http://www.mothur.org/wiki/Greengenes-f > … _databases ,about greengenes reference allignment, that it should be used (if necessary, but why!?) to align sequences. You also suggest not to use it for real analysis due to poor allignment. So, what can be used for allignment in place of this if one is using greengenes.

Do not use greengenes for alignments. You can use it for classification. Use silva for alignments and classification if you want.

  1. My second question is while using greengenes, how can I find start and end position of my sequences that has to be fed at pcr.seqs command line ?
  1. get an ecoli sequence and trim it to your primer positions
  2. align it against the greengenes alignment
  3. run summary.seqs on the aligned sequence
  4. use the start and end coordinates.

Hello Dr. Schloss,

I used recreated seed database files both for allignment and also for classification. I got better results. But I did not see Archaea in my files. I expect both Archaea and Bacteria in my samples because I used the primer sets that target both. kindly suggest me how to proceed, so that I can find a way.


looking forward,

Richa

Have you seen these pages?

http://blog.mothur.org/2014/08/08/SILVA-v119-reference-files/
http://www.mothur.org/wiki/Silva_reference_files

There are archaea in there.

Hello,

When I use recreated seed database reference files for classification, I get this kind of message in mothur and this step fails:

‘AB183858.UncB4157’ is in your template file and is not in your taxonomy file. Please correct.
‘HQ197980.HvnArane’ is in your template file and is not in your taxonomy file. Please correct.
‘L01575.ThyFlexu’ is in your template file and is not in your taxonomy file. Please correct.
‘AB282889.LacSimi2’ is in your template file and is not in your taxonomy file. Please correct.

-I used the same silva.seed_v119.align file for alignment.


please help and suggest.

looking forward.

Since you seem to be having problems with a lot of things across mothur, I would strongly encourage you to use files that we know work before you go off and try new things.