I was wondering how to modify the start and end numbers to use on the align.seqs command for fungal ITS1Fseq-ITS2seq primers and protist 18S V9Fseq-V9Rseq primers.
Hi there,
I’d encourage you to check out the blog post on this topic. For the 18S V9 region, you’ll have to find a reference 18S rRNA gene sequence that you can trim to the V9 region and then align. ITS is more tricky - these sequences are not homologous across organisms and so they do not lend themselves to a multiple sequence alignment. For ITS, I’d suggest using pairwise alignments like you can do in pairwise.seqs.
Hope this helps,
Pat
I am seeing mothur > pairwise.seqs(fasta=amazon.fasta, align=needleman). So, should I find a reference ITS FASTA file and replace it with amazon.fasta? Where can I get the reference FASTA file from? Or should I do something else?
I also found Saccharomyces cerevisiae as a reference 18S rRNA gene and trying to get the sequence. Should I use the FASTA sequence of yeast in the place like I am seeing for E. coli here?
E.coli
ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGT
GGCGGACGGGTGAGTAATGTCTGGGAAGCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCA
TAATGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTTGTTGG
TGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACG
GTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGT
ATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGAC
GTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGA
ATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCA
TCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGG
AGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGA
TTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCT
AACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAG
CGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAG
ATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTT
AAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGA
TAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGC
GCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAA
CTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTAC
ACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTT
TGTGATTCATGACTGGGGTG
And then find the reverse complement of the reverse primer sequence V9Rseq? After that should I run mothur > align.seqs(fasta=ecoli_v3.fasta, reference=silva.seed_v123.align)
mothur > summary.seqs(fasta=ecoli_v3.align) and replace ecoli_v3.fasta with yeast_v9.align but keep silva.seed_v123.align same? And finally do mothur > pcr.seqs(fasta=silva.seed_v123.align, start=6388, end=13861, keepdots=FALSE) but change the numbers at start and end before keepdots as it shows here? For ITSiFseq-ITS2seq, should I do the same? I am very new to MOTHUR so I am anxious and asking all these stupid questions. I would appreciate if you help me or post for ITS and 18S as well in the main website like you did for bact 16S.
You’d put your ITS file name in place of amazon.fasta
If you have a Saccharomyces cerevisiae 18S sequence, that would work in place of the E. coli seqeuence.
Pat
Hi Pat, Thank you for replying. You said to use my ITS file name in place of amazon.fasta for mothur > pairwise.seqs(fasta=amazon.fasta, align=needleman) command. Just curious, should this ITS file be a reference fasta file of a reference ITS fungal individual like Saccharomyces cerevisiae as a reference 18S rRNA gene?
Its impossible to generate a meaningful multiple sequence alignment of ITS sequences. So there’s no need to try to generate a reference alignment. It should be the file with the sequences you are analyzing.
Pat
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