How to set my reference sequences (sequence.keep)

[Miao Sun]

Hi All,

Anybody have any experience of how to set your reference sequences properly to run PHLAWD? I mean what's criterion of choosing them? Are they should be sequence representative or taxa  representative?

Should they be alignment or download them from GenBank should be fine? Any length required?

I'm really puzzled about abovementioned issues. Because it seems that it will cause "Segmentation fault" on PLAWD, if your reference sequences contain errors.

Thanks!

Miao

[Cody Hinchliff]

The segmentation fault suggests that your sequence contains illegal characters, like ? or -, etc. keep sequences should only contain nucleotide coding chars. Try removing everything except the letters A C G T from your keep sews and see if that solves your problem.

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[Miao Sun]

Thanks Cody!

Now things are becoming more weird. I don't think I have illegal characters in my reference sequences:

gi|54111867|gb|AY752474.1| Geranium sessiliflorum var. arenarium 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence

GGCGGTTCGCTGCCGGCGACGTCGCGAGAAGTCCACTGAACCTTATCATTTAGAGGAAGGAGAAGTCGTA

ACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTGTCGAACCCTGCACAGCAGAACGACCCGCGAAC

TCGTTAACAAACCGCGGGGAGCGGGTGGCGCTTGCGCCCCCCGCAACCCGATGTCGGGGGCTTGGGCGGA

AGCCCACGCTGCCTGACAAAAAAGTACCCCCGGCGCGGTCCGCGCCAAGGAATCGAAACGAAGCAATGTG

TGCCGTCCGCCCCGTTCGCGGGAAGTGGACGGCAACACGGTCTTCCAATGTATACTAAACGACTCTCGGC

AACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAAT

CCCGTGAACCATCGAGTTTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCCGAGGGCACGCCTGCCTGG

GCGTCACGCGCTCCGTCGCCCCGCAACCCCGAACCCCGAAACGGGCCAGGGTGCTTGTGGTGCGGACATT

GGTCTCCCGTGTGCCTTGCTCGCGGCTGGCCTAAAATTGAGTCCCGGACGCTCTGTTCTGCGGCCGACGG

TGGTTGAGAAGCCCTCGAAAACGTGCCGCTGCAGTGCTGCCATATGCGGACCCCTTGACCCTTGCGCGAC

CTCTCCCATTTGGGTGAGGGAGCTCCATCTGCGACCCCAGGTCAGGCGGGGCTACCCGCTGAATTTAAGC

ATATCAATAAG

>gi|124390016|gb|EF219365.1| Abutilon menziesii strain PCMB2763 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence

TGCGGAAGGATCATTGTCAGAAACCTGCCTAGCAGAACCACCCGTGAATGTGTTATCATACAAAACAGCG

AGAGGGTGCGGATGCAATATTGTGCCAACCCCTCTCGATGCCTTGGTGCGTTTGGTCTTGCCTCAACCCA

CCTCGTGTGGGTGAGCTGCAAGTTCCATCCACTCCAAGGCAAAACTAACAACCCCCGGCGCGAATTGCGC

CAAGGAATTTAAATRAAAAGAGTGCACGTCATTGTCGCCGACCCGTTCGCGGTGTTTGTGCGGGAGTGTC

GTAGCTAACTTTGTCGTGAAATACAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATAAAG

AACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCAAGTCTTTGAACGCAAGT

TGCGCCCCAAGCCATTAGGCCGAGGGCACGTCTGCCTGGGTGTCACGCATCGTTGCCCCAATCAAGCCTC

GAGCTTATTCTGCTCAGGTCAAATTAYGGGCGGATATTGGCTTCCCGTTCGCTCACCGTGCGCGGTTGGC

CTAAAAATGAGTCTTYGGCGATGAAGTGCCGCGACAATCGGTGGGAATACTTACAGTTGTCTCGTTTGAA

GTCGTGTGCACTYGTTGATTTAGACCCTATGACCCTTTTGGCATCACATCGTTGGTGCTCGCATCGCGAC

CCCAGGTCAGGCGGGAT

>gi|686985121|gb|KM037616.1| Rubus ulmifolius x Rubus caesius isolate UxC_col06 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence

TCCGTAGGTGAACCTGCGGAAGGATCATTGTCGAAACCTGCCCAGCAGAACGACCCGAGAACATGTTTCA

ACGCTTGGGGGCGAAGGGTCTTTCGGCTCCTCGTCCCTTTTCTCGGGAGGCAATCGTCTTGTGCGTTGCA

TCTCGATGCTCGCACTTGAACGACCCTCTCGGGCGTACAAACGAACACCGGCGTGTATTGCGCCAAGGAA

CTTGAATGAAAGAGCGTTCCCCCGCCGCCCCGGAAACGGTGTGCGTGCGGTGGGTTACGTCATCTTCAAT

ATGTCTAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATA

CTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGG

CCGAGGGCACGCCTGCCTGGGCGTCACACGTCGTTGCCCCCCCCCAAACCCCTCGGGAGTTGGGCGGGAC

GGATGATGGCCTCCCGTGTGCTCTGTCATGCGGTTGGCATAAAAACAAGTCCTCGGCGACTAACGCCACG

ACAATCGGTGGTTGTCAAACCTCTGTTGCCTATCGTGTGCGCGTGTCGAGCGAGGGCTCAACAAACCATG

TTGCATCGATTCGTCGATGCTTTCAACGCGACCCCAGGTCAGGCGGGGTTACCCGCTGAATTTAAGCATA

TCAATAAGCGGAGGA

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Dr. Miao Sun

Tel: 352 (284) 0928                 Email: cac...@ufl.edu

Florida Museum of Natural History, University of Florida
Dickinson Hall, 1659 Museum Rd，PO. Box 117800, Gainesville, FL 32611-7800, USA.

[Cody Hinchliff]

Try getting rid of the long Id strings. I would just use cut out everything except gi number and give it another try.

[Miao Sun]

Thanks, I'll redo it, and let you know the updates.

Miao

在 2015年2月19日星期四 UTC-5上午11:21:04，Cody Hinchliff写道：

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Dr. Miao Sun

Tel: 352 (284) 0928                 Email: cac...@ufl.edu

Florida Museum of Natural History, University of Florida
Dickinson Hall, 1659 Museum Rd，PO. Box 117800, Gainesville, FL 32611-7800, USA.

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[Miao Sun]

Hi,

I still come across the same "segmentation fault" (see the captured screen). 

Thanks!

Miao

在 2015年2月19日星期四 UTC-5上午11:21:04，Cody Hinchliff写道：

Try getting rid of the long Id strings. I would just use cut out everything except gi number and give it another try.

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Dr. Miao Sun

Tel: 352 (284) 0928                 Email: cac...@ufl.edu

Florida Museum of Natural History, University of Florida
Dickinson Hall, 1659 Museum Rd，PO. Box 117800, Gainesville, FL 32611-7800, USA.

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[Miao Sun]

Hi Cody,

I can't sleep for this. I've tried many times with different sequences with various modifications (e.g. remove all the tags, or remove part sequences, or change to "search = internal transcribed spacer"), and before I set them as reference sequences, I always make sure the sequences contain no other characters except ATCG.

So wonder:

1. how exactly then PLAWD work in side to capture the patterns of reference sequence? is it really strict or ITS evolves too fast that too much variables that made the procedure fail?

2. I think in Stephen's Mega-phylogeny (Smith, S. A., J. Beaulieu, and M. J. Donoghue. 2009.  Mega-phylogeny approach for comparative biology: an alternative to supertree and supermatrix approaches. BMC Evol Biol. . 9: 37), he used ITS right? How did he set the ITS reference sequences？Can I have his reference copy of ITS, just to make the PHLAWD work?

Thanks！

Miao