Hi,
My align.seqs is eliminating most of the basepairs. I know this problem is usually related to 5’->3’ vs. 3’->5’ sequencing, but I analyzed it both ways and the result remains the same (logfile attached below). Please help…
Windows version
Running 64Bit Version
mothur v.1.31.2
Last updated: 6/13/2013
by
Patrick D. Schloss
Department of Microbiology & Immunology
University of Michigan
pschloss@umich.edu
http://www.mothur.org
When using, please cite:
Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23):7537-41.
Distributed under the GNU General Public License
Type ‘help()’ for information on the commands that are available
Type ‘quit()’ to exit program
Interactive Mode
mothur > summary.seqs(fasta=COHEN_2538_831FB.fna)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 100 100 0 4 1
2.5%-tile: 1 124 124 0 4 5252
25%-tile: 1 270 270 0 4 52518
Median: 1 409 409 0 5 105036
75%-tile: 1 469 469 1 5 157553
97.5%-tile: 1 532 532 3 7 204819
Maximum: 1 673 673 10 20 210070
Mean: 1 368.258 368.258 0.378826 4.87502
of Seqs: 210070
Output File Names:
COHEN_2538_831FB.summary
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.
mothur > trim.seqs(fasta=COHEN_2538_831FB.fna, oligos=bdellovibrio.oligos, qfile=COHEN_2538_831FB.qual, maxambig=0, maxhomop=8, bdiffs=1, pdiffs=2, minlength=150, qwindowaverage=30, qwindowsize=50, processors=8)
Using 8 processors.
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Appending files from process 1
Appending files from process 2
Appending files from process 3
Appending files from process 4
Appending files from process 5
Appending files from process 6
Appending files from process 7
Group count:
1-831F 2619
10-831F 4113
10B-831F 3490
11-831F 10493
11B-831F 7701
12-831F 2669
12B-831F 2365
17-831F 2105
18-831F 4039
19-831F 1205
1B-831F 3399
2-831F 3884
20-831F 1435
21-831F 1426
22-831F 3419
23-831F 7062
24-831F 893
2B-831F 10364
3-831F 2114
3B-831F 5413
3ES1-831F 354
3ES2-831F 3903
4-831F 2156
4B-831F 3267
5-831F 6068
5B-831F 3011
5K1-831F 11535
5K2-831F 4916
6-831F 1771
6B-831F 2683
7-831F 2546
7B-831F 6392
8-831F 8042
8B-831F 2386
9-831F 2876
9B-831F 1390
Total of all groups is 143504
Output File Names:
COHEN_2538_831FB.trim.fasta
COHEN_2538_831FB.scrap.fasta
COHEN_2538_831FB.trim.qual
COHEN_2538_831FB.scrap.qual
COHEN_2538_831FB.groups
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.
mothur > summary.seqs(fasta=COHEN_2538_831FB.trim.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 150 150 0 2 1
2.5%-tile: 1 161 161 0 3 3588
25%-tile: 1 246 246 0 4 35877
Median: 1 325 325 0 5 71753
75%-tile: 1 393 393 0 5 107629
97.5%-tile: 1 455 455 0 7 139917
Maximum: 1 551 551 0 8 143504
Mean: 1 316.687 316.687 0 4.67473
of Seqs: 143504
Output File Names:
COHEN_2538_831FB.trim.summary
mothur > unique.seqs(fasta=Cohen_2538_831FB.trim.fasta) [WARNING]: This command can take a namefile and you did not provide one. The current namefile is Cohen_2538_831FB.trim.names which seems to match Cohen_2538_831FB.trim.fasta. 1000 840 2000 1417 3000 2106 4000 2742 5000 3368 6000 4012 7000 4728 8000 5070 9000 5812 10000 6383 11000 7135 12000 7793 13000 8501 14000 9218 15000 9962 16000 10596 17000 11321 18000 12102 19000 12854 20000 13477 21000 14223 22000 14792 23000 15526 24000 15895 25000 16719 26000 17318 27000 17930 28000 18402 29000 19045 30000 19675 31000 20485 32000 21105 33000 21740 34000 22557 35000 23216 36000 23926 37000 24277 38000 24756 39000 25247 40000 26003 41000 26815 42000 27395 43000 27958 44000 28841 45000 29670 46000 30217 47000 31023 48000 31509 49000 32236 50000 32889 51000 33544 52000 34042 53000 34598 54000 35194 55000 35709 56000 36125 57000 36795 58000 37175 59000 37940 60000 38663 61000 39265 62000 39749 63000 40233 64000 40827 65000 41449 66000 42005 67000 42534 68000 43056 69000 43779 70000 44142 71000 44777 72000 45331 73000 45751 74000 46287 75000 46679 76000 47063 77000 47562 78000 48036 79000 48706 80000 49433 81000 49995 82000 50726 83000 51392 84000 51893 85000 52428 86000 52754 87000 53330 88000 53797 89000 54353 90000 54913 91000 55482 92000 55994 93000 56316 94000 56954 95000 57518 96000 57820 97000 58034 98000 58242 99000 58722 100000 59360 101000 59744 102000 59865 103000 59969 104000 60055 105000 60137 106000 60226 107000 60869 108000 61311 109000 61762 110000 62406 111000 62950 112000 63680 113000 64318 114000 65005 115000 65644 116000 66236 117000 66896 118000 67462 119000 67928 120000 68403 121000 68924 122000 69175 123000 69642 124000 70066 125000 70445 126000 70697 127000 70967 128000 71535 129000 72060 130000 72662 131000 73156 132000 73625 133000 74238 134000 74740 135000 75385 136000 75820 137000 76207 138000 76494 139000 76868 140000 77468 141000 77906 142000 78336 143000 78719 143504 79078
Output File Names:
Cohen_2538_831FB.trim.names
Cohen_2538_831FB.trim.unique.fasta
mothur > summary.seqs(fasta=Cohen_2538_831FB.trim.unique.fasta, name=Cohen_2538_831FB.trim.names)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 150 150 0 2 1
2.5%-tile: 1 161 161 0 3 3588
25%-tile: 1 246 246 0 4 35877
Median: 1 325 325 0 5 71753
75%-tile: 1 393 393 0 5 107629
97.5%-tile: 1 455 455 0 7 139917
Maximum: 1 551 551 0 8 143504
Mean: 1 316.687 316.687 0 4.67473
of unique seqs: 79078
total # of seqs: 143504
Output File Names:
Cohen_2538_831FB.trim.unique.summary
mothur > align.seqs(fasta=COHEN_2538_831FB.trim.unique.fasta, reference=silva.bacteria.fasta, flip=T, processors=8)
Using 8 processors.
Reading in the silva.bacteria.fasta template sequences… DONE.
It took 69 to read 14956 sequences.
Aligning sequences from COHEN_2538_831FB.trim.unique.fasta …
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences… DONE.
It took 871 to read 14956 sequences.
DONE.
It took 1001 to read 14956 sequences.
DONE.
It took 1002 to read 14956 sequences.
DONE.
It took 1040 to read 14956 sequences.
DONE.
It took 1054 to read 14956 sequences.
DONE.
It took 1139 to read 14956 sequences.
DONE.
It took 1408 to read 14956 sequences.
Some of you sequences generated alignments that eliminated too many bases, a list is provided in COHEN_2538_831FB.trim.unique.flip.accnos. If the reverse compliment proved to be better it was reported.
It took 6196 secs to align 79078 sequences.
Output File Names: COHEN_2538_831FB.trim.unique.align COHEN_2538_831FB.trim.unique.align.report COHEN_2538_831FB.trim.unique.flip.accnos
mothur > summary.seqs(fasta=COHEN_2538_831FB.trim.unique.align, name=Cohen_2538_831FB.trim.names)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 1044 1046 2 0 1 3588
25%-tile: 1044 1071 7 0 2 35877
Median: 43026 43116 11 0 3 71753
75%-tile: 43097 43116 18 0 3 107629
97.5%-tile: 43115 43116 45 0 5 139917
Maximum: 43116 43116 433 0 8 143504
Mean: 26437.7 26493.6 13.7358 0 2.55593
of unique seqs: 79078
total # of seqs: 143504
Output File Names:
COHEN_2538_831FB.trim.unique.summary
mothur > align.seqs(fasta=COHEN_2538_831FB.trim.unique.fasta, reference=silva.bacteria.fasta, processors=8)
Using 8 processors.
Reading in the silva.bacteria.fasta template sequences… DONE.
It took 71 to read 14956 sequences.
Aligning sequences from COHEN_2538_831FB.trim.unique.fasta …
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences… DONE.ique.
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences… DONE.ique.
It took 735 to read 14956 sequences. It took 735 to read 14956 sequences. It took 735 to read 14956 sequences. DONE. DONE.DONE.It took 735 to read 14956 sequences. DONE.
It took 736 to read 14956 sequences.
It took 736 to read 14956 sequences.
It took 736 to read 14956 sequences.
Some of you sequences generated alignments that eliminated too many bases, a list is provided in COHEN_2538_831FB.trim.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well.
It took 4015 secs to align 79078 sequences.
Output File Names: COHEN_2538_831FB.trim.unique.align COHEN_2538_831FB.trim.unique.align.report COHEN_2538_831FB.trim.unique.flip.accnos
mothur > summary.seqs(fasta=COHEN_2538_831FB.trim.unique.align, name=Cohen_2538_831FB.trim.names)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: -1 -1 0 0 1 3588
25%-tile: 1044 1058 2 0 1 35877
Median: 43026 43116 5 0 2 71753
75%-tile: 43112 43116 12 0 3 107629
97.5%-tile: 43116 43116 34 0 4 139917
Maximum: 43116 43116 302 0 8 143504
Mean: 24049.6 24079.1 8.6797 0 1.91124
of unique seqs: 79078
total # of seqs: 143504
Output File Names:
COHEN_2538_831FB.trim.unique.summary
mothur > trim.seqs(fasta=COHEN_2538_831FB.fna, oligos=bdellovibrio.oligos, qfile=COHEN_2538_831FB.qual, maxambig=0, maxhomop=8, bdiffs=1, pdiffs=2, minlength=150, qwindowaverage=30, qwindowsize=50, flip=T, processors=8)
Using 8 processors.
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Appending files from process 1
Appending files from process 2
Appending files from process 3
Appending files from process 4
Appending files from process 5
Appending files from process 6
Appending files from process 7
Group count:
1-831F 2619
10-831F 4113
10B-831F 3490
11-831F 10493
11B-831F 7701
12-831F 2669
12B-831F 2365
17-831F 2105
18-831F 4039
19-831F 1205
1B-831F 3399
2-831F 3884
20-831F 1435
21-831F 1426
22-831F 3419
23-831F 7062
24-831F 893
2B-831F 10364
3-831F 2114
3B-831F 5413
3ES1-831F 354
3ES2-831F 3903
4-831F 2156
4B-831F 3267
5-831F 6068
5B-831F 3011
5K1-831F 11535
5K2-831F 4916
6-831F 1771
6B-831F 2683
7-831F 2546
7B-831F 6392
8-831F 8042
8B-831F 2386
9-831F 2876
9B-831F 1390
Total of all groups is 143504
Output File Names:
COHEN_2538_831FB.trim.fasta
COHEN_2538_831FB.scrap.fasta
COHEN_2538_831FB.trim.qual
COHEN_2538_831FB.scrap.qual
COHEN_2538_831FB.groups
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.
mothur > summary.seqs(fasta=COHEN_2538_831FB.trim.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 150 150 0 2 1
2.5%-tile: 1 161 161 0 3 3588
25%-tile: 1 246 246 0 4 35877
Median: 1 325 325 0 5 71753
75%-tile: 1 393 393 0 5 107629
97.5%-tile: 1 455 455 0 7 139917
Maximum: 1 551 551 0 8 143504
Mean: 1 316.687 316.687 0 4.67473
of Seqs: 143504
Output File Names:
COHEN_2538_831FB.trim.summary
mothur > unique.seqs(fasta=Cohen_2538_831FB.trim.fasta) [WARNING]: This command can take a namefile and you did not provide one. The current namefile is Cohen_2538_831FB.trim.names which seems to match Cohen_2538_831FB.trim.fasta. 1000 840 2000 1417 3000 2106 4000 2742 5000 3368 6000 4012 7000 4728 8000 5070 9000 5812 10000 6383 11000 7135 12000 7793 13000 8501 14000 9218 15000 9962 16000 10596 17000 11321 18000 12102 19000 12854 20000 13477 21000 14223 22000 14792 23000 15526 24000 15895 25000 16719 26000 17318 27000 17930 28000 18402 29000 19045 30000 19675 31000 20485 32000 21105 33000 21740 34000 22557 35000 23216 36000 23926 37000 24277 38000 24756 39000 25247 40000 26003 41000 26815 42000 27395 43000 27958 44000 28841 45000 29670 46000 30217 47000 31023 48000 31509 49000 32236 50000 32889 51000 33544 52000 34042 53000 34598 54000 35194 55000 35709 56000 36125 57000 36795 58000 37175 59000 37940 60000 38663 61000 39265 62000 39749 63000 40233 64000 40827 65000 41449 66000 42005 67000 42534 68000 43056 69000 43779 70000 44142 71000 44777 72000 45331 73000 45751 74000 46287 75000 46679 76000 47063 77000 47562 78000 48036 79000 48706 80000 49433 81000 49995 82000 50726 83000 51392 84000 51893 85000 52428 86000 52754 87000 53330 88000 53797 89000 54353 90000 54913 91000 55482 92000 55994 93000 56316 94000 56954 95000 57518 96000 57820 97000 58034 98000 58242 99000 58722 100000 59360 101000 59744 102000 59865 103000 59969 104000 60055 105000 60137 106000 60226 107000 60869 108000 61311 109000 61762 110000 62406 111000 62950 112000 63680 113000 64318 114000 65005 115000 65644 116000 66236 117000 66896 118000 67462 119000 67928 120000 68403 121000 68924 122000 69175 123000 69642 124000 70066 125000 70445 126000 70697 127000 70967 128000 71535 129000 72060 130000 72662 131000 73156 132000 73625 133000 74238 134000 74740 135000 75385 136000 75820 137000 76207 138000 76494 139000 76868 140000 77468 141000 77906 142000 78336 143000 78719 143504 79078
Output File Names:
Cohen_2538_831FB.trim.names
Cohen_2538_831FB.trim.unique.fasta
mothur > align.seqs(fasta=COHEN_2538_831FB.trim.unique.fasta, reference=silva.bacteria.fasta, flip=T, processors=8)
Using 8 processors.
Reading in the silva.bacteria.fasta template sequences… DONE.
It took 73 to read 14956 sequences.
Aligning sequences from COHEN_2538_831FB.trim.unique.fasta …
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences… Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences…
Reading in the silva.bacteria.fasta template sequences... Reading in the silva.bacteria.fasta template sequences... Reading in the silva.bacteria.fasta template sequences... DONE. It took 823 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. DONE. It took 824 to read 14956 sequences. Some of you sequences generated alignments that eliminated too many bases, a list is provided in COHEN_2538_831FB.trim.unique.flip.accnos. If the reverse compliment proved to be better it was reported. It took 5847 secs to align 79078 sequences.
Output File Names: COHEN_2538_831FB.trim.unique.align COHEN_2538_831FB.trim.unique.align.report COHEN_2538_831FB.trim.unique.flip.accnos
mothur > summary.seqs(fasta=COHEN_2538_831FB.trim.unique.align, name=Cohen_2538_831FB.trim.names)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 1044 1046 2 0 1 3588
25%-tile: 1044 1071 7 0 2 35877
Median: 43026 43116 11 0 3 71753
75%-tile: 43097 43116 18 0 3 107629
97.5%-tile: 43113 43116 45 0 5 139917
Maximum: 43116 43116 433 0 8 143504
Mean: 26278.8 26334.7 13.7358 0 2.56091
of unique seqs: 79078
total # of seqs: 143504
Output File Names:
COHEN_2538_831FB.trim.unique.summary