I recently amplified two genes (plastid 16s and ITS1-4) from an unknown algae. The sequencing results were:
plastid 16s rRNA
CACCCCAGTCATCAGCTCTACCTTAGGCGAACCCTTTCAGGTAACGACTTCGGGCATAACCAACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGAATTCACCGCCGTATTGCTGACCGGCGATTACTAGCGATTCCAGCTTCATGCAGGCGAGTTTCAGCCTGCAATCCGAACTTGGACTAAGTTTCGGAGATTTGCTAACTTTCGCAAGCTTGCTTCTCATTGTCTTAGTCATTGTAGCACGTGTGTAGCCCAGGATATAAGGGGCATGCTGACTTGACGTCATCCTTACCTTCCTCCCGCTTTTCACGGGCAGTATCTCTATAGTCCCCCACTTAAGAATGGCAAATAAAGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTATCTCGGCTCCCGAAGGCACTTTCTCTATTATAAGAAATTCCGAGTATGTCAAATCCTGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACACTTGCGTGCGTACTCCCCAGGCGGGATACTTAACGCGTTAGCTACGACACCGCAGGTAAACCCGCAACATCTAGTATCCATCGTTTACGGCTAGGACTACCGGGGTATCTAATCCCGTTTGCTACCCTAGCTTTCGTCTCTGAGTGTCAGTAATGGCCCAGTAGGACGCCTTCGCTAATGGTGTTCTTCCTAATATCTACGCATTTCACCGCTACACTAGAAATTCCTCCTACCCCTACCATACTCAAGTCTAGCAGTTTCTGCTGCACTCCTGAAGTTAAGCTTCAGTCTTTAACGGCAGACTTACTAAACAACCTGCAGACGCTTTACGCCCAGTGATTCCGGATAACGCTCGCATCTCCCGTATTACCGCGGCTGCTGGCACGGAATTAGCCGATGCTTATTCCCCAAGTACCGTCAGAACTTCTTCCTTGGGAAAAGAGGTTTACGACCCACGAGCCTTCATCCCTCACGCGGTATTGCTCCGTCAGGCTTTCGCCCATTGCGGAAAATTCCCCACTGCTGCCTCCCGGAGGAGTCTGGGCCGTATCTCAGTCCCAGTGTGGCTGCTCATCCTCTCAAACCAGCTACTGATCGTGGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATCAGGCGCGAGCCCATCTCCAGGCAGATTATTAAATCTATTTCACCCTTAGGCATATGGGGCTTTTTATACCCCTCCTAGAGGTAGGTTCTCACGTTATACTCACCCGTTCGCTACTCAAATCTATAAAGATTCGCGTTCAACTTGCATGTGTTAGGCATACCGCCAGCGTTCATC
ITS1-4
TCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTCGCCTTCTCTGAGTTGAGGCCTTGCCTTCAAGGTGAAATTCCTCTCGCCTTTACACGTAGTATACACTTGCTGTGAAAAGAAAAGCCAAGCAAGCCTCACAGCACACACCGATTGAGGAAATCAATCCTTACCACGGTGCGGCGCATGAGGAATGACCCTCCCCGCAAATGCTCCACTTGAGACGACGCGCAACTCTTTAACGAGCCACCCGACGCCCACGCTCCGCAAACCGTCCGTGAACGGTGCGATGTGGGGGGAACGGCACTCAACCAGGGATACTTCCAGGCAGAACCTGGAAGTGCAAGATGCGTTCAAAGATTTGATGATTCACTGAATCCTGCAATTCGCACTACGTATCGCATTTCGCTGCGTTCTTCATCGTTGCGAGAGCCAAGACATTCGTTGTTGGGAGTTGTCTTTGCTTTCTTTGTACAGACACACTACGTGCAAGCGCGCAGAAGCGCGCACACACACAGGTGTGACCGCACGACCGGCCCTACAAAGAGGCGCGGCCATCGCCCCGCAGAATCAACCGCGGGACCGAGAGGTGAAACACGCGTGTGCGAGAAGAAGCGCCCAAAACCCCTGATGCAAACATCAGAGACAAAGGACGCTCCCAAAAGACGAGCACGGTGATACACGGTGGGTATGTGGATTATTCGGTAATGATCCTTCCGCAGGTTCACCTACGGA
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For plastid 16s rRNA, the nblast results
1. Rebecca salina Query (100%), E-value (0), ident (97%)
2.Exanthemachrysis gayraliae Query (100%), E-value (0), ident (96%)
3,4,5,6,7,8,9,10
I have selected the top 10 species and downloaded their FASTA (alignment sequence) from nblast. I used Mega6 to perform multiple alignment, following evolutionary tree construction by neighbour-joining, maximum likelihood, bootstrap (1000 replicates). However, the tree result showed that my unknown of algae was out-group (enclosed file). I wondered it is a correct phylogenetic tree.
Besides, the nblast results of ITS1-4 sequence showed poor query %, e-value and ident %.
So, I am pleased to have your suggestions. Thank you.
If you have poor similarity of sequences, of curse you wouldn't able to construct phylogeny. possible reasons: 1. you amplify wrong sequence (not ITS)?
2. sequence is in complementary direction (check alignment in the blast- strand is positive or negative; if negative, use sequence reverse compliment online tool);
3. there is no sequence of that organism in the data base.
so, I checked, your seq is Rebecca salina and is in "minus strand". But Mega6 could not convert your sequence to reverse compliment. Therefore first, convert it with: http://www.bioinformatics.org/sms/rev_comp.html and use reverse compliment to further alignment. Before constructing phylogeny, crop your sequence and all database sequence to equal sequence region.
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