Hi,
I was on the Detroit course a few weeks ago. Since then I’ve been trying to use mothur on some PacBio reads. I have an error message come up as follows: [ERROR]: Your name file contains 182091 valid sequences, and your groupfile contains 191161, please correct.
I’ve tried so many ways of correcting this, but it still happens. I had to use unique.seqs to make a name file. I’ve pasted the log below, any suggestions would be greatly appreciated!
Thanks so much,
Bethan
mothur v.1.30.2
Last updated: 4/19/2013
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
mothur > summary.seqs(fasta=Antarctic1.fasta)
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 20 20 0 2 1
2.5%-tile: 1 413 413 0 3 5039
25%-tile: 1 497 497 0 4 50389
Median: 1 500 500 0 5 100777
75%-tile: 1 502 502 0 5 151165
97.5%-tile: 1 992 992 0 7 196514
Maximum: 1 2102 2102 0 24 201552
Mean: 1 514.994 514.994 0 4.65406
of Seqs: 201552
Output File Names:
Antarctic1.summary
mothur > trim.seqs(fasta=current, maxhomop=8, minlength=413, maxlength=650, flip=T)
Using Antarctic1.fasta as input file for the fasta parameter.
Using 1 processors.
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Output File Names: Antarctic1.trim.fasta Antarctic1.scrap.fasta
mothur > summary.seqs()
Using Antarctic1.trim.fasta as input file for the fasta parameter.
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 413 413 0 3 1
2.5%-tile: 1 477 477 0 3 4813
25%-tile: 1 497 497 0 4 48121
Median: 1 500 500 0 5 96242
75%-tile: 1 502 502 0 5 144362
97.5%-tile: 1 524 524 0 7 187670
Maximum: 1 650 650 0 8 192482
Mean: 1 499.152 499.152 0 4.60035
of Seqs: 192482
Output File Names:
Antarctic1.trim.summary
mothur > unique.seqs(fasta=current)
Using Antarctic1.trim.fasta as input file for the fasta parameter.
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Output File Names:
Antarctic1.trim.names
Antarctic1.trim.unique.fasta
mothur > align.seqs(fasta=current, group=mergegroups, reference=silva.eukarya.fasta, flip=T)
Using Antarctic1.trim.unique.fasta as input file for the fasta parameter.
group is not a valid parameter.
The valid parameters are: reference, fasta, search, ksize, match, align, mismatch, gapopen, gapextend, processors, flip, save, threshold, inputdir, and outputdir.
Using 1 processors.
[ERROR]: did not complete align.seqs.
mothur > align.seqs(fasta=current, reference=silva.eukarya.fasta, flip=T)
Using Antarctic1.trim.unique.fasta as input file for the fasta parameter.
Using 1 processors.
Reading in the silva.eukarya.fasta template sequences… DONE.
It took 3 to read 1238 sequences.
Aligning sequences from Antarctic1.trim.unique.fasta …
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Some of you sequences generated alignments that eliminated too many bases, a list is provided in Antarctic1.trim.unique.flip.accnos. If the reverse compliment proved to be better it was reported.
It took 2778 secs to align 128212 sequences.
Output File Names: Antarctic1.trim.unique.align Antarctic1.trim.unique.align.report Antarctic1.trim.unique.flip.accnos
mothur > get.current()
Current files saved by mothur: fasta=Antarctic1.trim.unique.align name=Antarctic1.trim.names
mothur > summary.seqs()
Using Antarctic1.trim.unique.align as input file for the fasta parameter.
Using 1 processors.
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is Antarctic1.trim.names which seems to match Antarctic1.trim.unique.align.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1044 1048 3 0 1 1
2.5%-tile: 34102 43116 460 0 3 3206
25%-tile: 34102 43116 477 0 4 32054
Median: 34102 43116 480 0 5 64107
75%-tile: 34102 43116 483 0 5 96160
97.5%-tile: 34105 43116 505 0 7 125007
Maximum: 43113 43116 622 0 8 128212
Mean: 34096 43102.3 480.31 0 4.6038
of Seqs: 128212
Output File Names:
Antarctic1.trim.unique.summary
mothur > get.current()
Current files saved by mothur: fasta=Antarctic1.trim.unique.align name=Antarctic1.trim.names
mothur > screen.seqs(fasta=current, name=current, group=mergegroups, end=43116, start=optimize, criteria=95, processors=2)
Using Antarctic1.trim.unique.align as input file for the fasta parameter.
Using Antarctic1.trim.names as input file for the name parameter.
[ERROR]: cannot convert optimize to an integer.
Using 2 processors.
[ERROR]: did not complete screen.seqs.
mothur > screen.seqs(fasta=current, name=current, group=mergegroups, end=43116, optimize=start, criteria=95, processors=2)
Using Antarctic1.trim.unique.align as input file for the fasta parameter.
Using Antarctic1.trim.names as input file for the name parameter.
Using 2 processors.
Optimizing start to 34104.
Processing sequence: 100
Processing sequence: 100
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Processing sequence: 200
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…
Processing sequence: 63200
Processing sequence: 63300
Processing sequence: 63400
Processing sequence: 63500
Processing sequence: 63600
Processing sequence: 63700
Processing sequence: 63800
Processing sequence: 63900
Processing sequence: 64000
Processing sequence: 64100
Processing sequence: 64107
Output File Names:
Antarctic1.trim.unique.good.align
Antarctic1.trim.unique.bad.accnos
Antarctic1.trim.good.names
mergegroupsgood
It took 1260 secs to screen 128212 sequences.
mothur > summary.seqs()
Using Antarctic1.trim.unique.good.align as input file for the fasta parameter.
Using 2 processors.
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is Antarctic1.trim.good.names which seems to match Antarctic1.trim.unique.good.align.
m
Start End NBases Ambigs Polymer NumSeqs
Minimum: 28432 43116 415 0 3 1
2.5%-tile: 34102 43116 468 0 3 3015
25%-tile: 34102 43116 478 0 4 30143
Median: 34102 43116 481 0 5 60285
75%-tile: 34102 43116 483 0 5 90427
97.5%-tile: 34104 43116 505 0 7 117554
Maximum: 34104 43116 622 0 8 120568
Mean: 34095.9 43116 481.749 0 4.56966
of Seqs: 120568
Output File Names:
Antarctic1.trim.unique.good.summary
mothur > filter.seqs(fasta=current, vertical=T, trump=., processors=2)
Using Antarctic1.trim.unique.good.align as input file for the fasta parameter.
Using 2 processors.
Creating Filter…
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50000
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50100
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50100
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50900
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51100
51000
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51100
51200
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51500
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51500
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51700
51900
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52000
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52100
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52100
52300
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53000
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56000
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55800
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55900
56400
56000
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56100
56600
56200
56700
56300
56800
56400
56900
56500
57000
56600
57100
56700
57200
56800
57300
56900
57400
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57800
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57900
57600
58000
58100
57700
58200
57800
58300
57900
58400
58000
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58200
58600
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58300
58800
58400
58900
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59000
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59700
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59800
59500
59900
59600
60000
59700
60100
59800
60200
59900
60285
60000
60100
60200
60283
Running Filter... 100 100 200 200 300 300 400 400 500 500 600 600 700 700 800 800 900 900 1000 1000 1100 1100 1200 1300 1200 1400 1300 1500 1400 1500 1600 1600 1700 1700 1800 1800 1900 1900 2000 2000 2100 2100 2200 2200 2300 2400 2300 2400 2500 2500 2600 2600 2700 2700 2800 2800 2900 2900 3000 3000 3100 3200 3100 3300 3200 3400 3300 3500 3400 3600 3700 3500 3800 3600 3900 3700 4000 3800 4100 3900 4200 4300 4000 4400 4100 4200 4500 4300 4600 4700 4400 4800 4500 4900 5000 4600 5100 4700 5200 4800 5300 5400 4900 5500 5000 5600 5100 5700 5200 5800 5300 5900 5400 6000 5500 6100 5600 6200 6300 5700 6400 5800 6500 5900 6600 6000 6100 6700 6800 6200 6900 6300 7000 7100 6400 7200 6500 7300 6600 7400 6700 7500 6800 7600 6900 7700 7000 7800 7100 7900 7200 8000 7300 7400 8100 8200 7500 7600 8300 8400 7700 8500 7800 8600 7900 8700 8000 8800 8100 8900 8200 9000 9100 9200 8300 8400 9300 8500 9400 8600 9500 8700 9600 8800 9700 9800 8900 9900 9000 10000 10100 9100 10200 9200 10300 9300 10400 9400 10500 9500 10600 9600 10700 10800 9700 10900 9800 9900 11000 10000 11100 11200 10100 11300 10200 10300 11400 11500 10400 11600 11700 10500 11800 10600 11900 10700 12000 10800 12100 10900 12200 11000 11100 12300 12400 12500 11200 12600 11300 12700 11400 12800 11500 11600 12900 11700 13000 13100 11800 11900 13200 12000 13300 12100 12200 13400 13500 12300 12400 13600 13700 12500 13800 12600 12700 13900 12800 14000 12900 14100 14200 13000 14300 13100 14400 13200 14500 13300 13400 14600 14700 13500 14800 13600 14900 13700 15000 13800 15100 13900 15200 14000 14100 15300 14200 15400 14300 15500 14400 15600 14500 14600 15700 15800 14700 14800 15900 14900 16000 15000 16100 15100 16200 15200 16300 15300 16400 15400 15500 16500 15600 16600 16700 15700 16800 15800 16900 15900 17000 17100 16000 17200 16100 17300 16200 17400 16300 17500 16400 17600 16500 17700 16600 17800 16700 17900 16800 18000 18100 16900 18200 17000 18300 17100 18400 17200 18500 17300 18600 17400 18700 17500 18800 18900 17600 19000 17700 19100 17800 19200 17900 18000 19300 18100 19400 18200 19500 18300 19600 18400 19700 19800 18500 19900 18600 20000 18700 20100 20200 18800 20300 18900 20400 19000 19100 20500 20600 19200 20700 19300 19400 20800 19500 20900 19600 19700 21000 19800 21100 19900 21200 20000 21300 20100 21400 20200 21500 20300 21600 21700 20400 21800 20500 20600 21900 20700 22000 22100 20800 22200 20900 22300 21000 22400 21100 22500 21200 22600 21300 22700 21400 22800 21500 22900 21600 23000 21700 23100 21800 23200 23300 21900 23400 22000 23500 22100 22200 23600 22300 23700 22400 23800 22500 23900 24000 22600 22700 22800 24100 24200 22900 24300 23000 24400 23100 24500 23200 23300 24600 23400 24700 23500 24800 23600 23700 24900 25000 23800 25100 25200 23900 24000 24100 25300 24200 25400 24300 25500 24400 25600 24500 25700 24600 25800 24700 25900 26000 24800 24900 26100 25000 26200 25100 25200 26300 26400 25300 26500 25400 26600 25500 26700 25600 26800 25700 25800 26900 25900 27000 27100 26000 26100 27200 26200 27300 26300 27400 26400 27500 26500 27600 26600 27700 26700 27800 26800 27900 28000 26900 28100 27000 28200 27100 28300 27200 28400 27300 28500 27400 28600 28700 27500 28800 28900 27600 29000 27700 29100 27800 29200 27900 29300 28000 29400 28100 29500 28200 29600 28300 29700 29800 28400 29900 28500 30000 28600 28700 30100 28800 30200 28900 30300 29000 30400 29100 30500 29200 30600 30700 29300 30800 29400 30900 29500 31000 29600 31100 29700 31200 29800 31300 29900 30000 31400 31500 30100 30200 30300 31600 30400 31700 31800 30500 31900 32000 30600 30700 32100 30800 32200 30900 32300 31000 32400 32500 31100 32600 32700 31200 32800 31300 32900 31400 31500 33000 33100 31600 33200 31700 33300 31800 33400 31900 33500 33600 32000 33700 32100 32200 32300 33800 32400 33900 32500 32600 32700 34000 32800 34100 34200 32900 34300 33000 33100 34400 33200 33300 34500 33400 34600 33500 33600 34700 33700 34800 33800 34900 33900 35000 34000 35100 34100 35200 34200 34300 35300 35400 34400 35500 35600 34500 35700 34600 35800 34700 35900 34800 36000 34900 36100 36200 35000 35100 36300 35200 35300 36400 35400 35500 36500 35600 36600 35700 36700 35800 36800 35900 36900 36000 37000 36100 37100 36200 37200 36300 37300 37400 36400 36500 37500 37600 36600 36700 37700 36800 37800 36900 37900 38000 37000 38100 37100 37200 38200 37300 38300 37400 38400 37500 38500 37600 38600 37700 38700 37800 38800 37900 38900 39000 38000 39100 38100 39200 38200 38300 39300 38400 39400 38500 39500 38600 39600 38700 39700 38800 39800 38900 39900 39000 40000 39100 40100 40200 39200 39300 40300 39400 40400 40500 39500 40600 39600 39700 40700 39800 40800 39900 40000 40900 40100 41000 40200 41100 40300 41200 40400 40500 41300 40600 41400 40700 41500 40800 41600 40900 41700 41000 41800 41100 41900 41200 42000 41300 42100 41400 42200 41500 42300 41600 42400 42500 41700 42600 41800 42700 41900 42800 42000 42100 42900 42200 43000 43100 42300 43200 42400 42500 43300 42600 42700 43400 42800 43500 43600 42900 43700 43000 43800 43100 43200 43900 44000 43300 44100 43400 44200 43500 44300 44400 43600 44500 43700 44600 43800 44700 43900 44800 44000 44900 44100 45000 44200 45100 44300 45200 44400 45300 44500 45400 44600 45500 44700 45600 44800 45700 45800 44900 45000 45900 45100 46000 46100 45200 46200 45300 46300 45400 46400 45500 46500 45600 46600 45700 46700 45800 46800 45900 46900 47000 46000 47100 46100 47200 46200 47300 46300 47400 46400 47500 46500 47600 46600 47700 46700 47800 47900 46800 48000 48100 46900 47000 48200 47100 48300 47200 48400 47300 47400 48500 47500 48600 47600 48700 47700 48800 47800 48900 47900 49000 49100 48000 49200 48100 48200 49300 49400 48300 48400 49500 49600 48500 49700 48600 49800 49900 48700 50000 48800 50100 50200 48900 49000 50300 49100 50400 49200 49300 50500 50600 49400 49500 50700 49600 50800 50900 49700 49800 51000 49900 51100 50000 51200 50100 51300 51400 50200 51500 50300 51600 51700 50400 51800 50500 51900 50600 50700 52000 50800 52100 50900 52200 52300 51000 52400 51100 52500 51200 52600 51300 52700 51400 52800 52900 51500 53000 51600 53100 53200 51700 53300 53400 51800 53500 51900 52000 53600 52100 53700 52200 53800 52300 53900 54000 52400 54100 54200 52500 54300 52600 52700 54400 52800 54500 52900 54600 53000 54700 53100 53200 54800 54900 53300 55000 53400 55100 53500 53600 55200 53700 55300 53800 55400 53900 55500 54000 55600 55700 54100 55800 54200 54300 54400 55900 54500 54600 56000 56100 54700 56200 54800 56300 56400 54900 56500 55000 56600 55100 56700 56800 55200 55300 56900 55400 57000 57100 55500 55600 57200 55700 55800 57300 55900 57400 57500 56000 56100 57600 57700 56200 57800 56300 57900 56400 56500 58000 56600 58100 56700 58200 56800 58300 56900 57000 58400 57100 58500 57200 58600 57300 58700 57400 58800 57500 57600 58900 57700 59000 57800 59100 57900 59200 58000 58100 59300 58200 59400 58300 59500 58400 59600 58500 59700 58600 59800 58700 59900 58800 60000 58900 60100 60200 59000 60285 59100 59200 59300 59400 59500 59600 59700 59800 59900 60000 60100 60200 60283
Length of filtered alignment: 1326 Number of columns removed: 48674 Length of the original alignment: 50000 Number of sequences used to construct filter: 120568
Output File Names:
Antarctic1.filter
Antarctic1.trim.unique.good.filter.fasta
mothur > get.current()
Current files saved by mothur: fasta=Antarctic1.trim.unique.good.filter.fasta group=mergegroupsgood name=Antarctic1.trim.good.names processors=2
mothur > unique.seqs(fasta=current, name=current)
Using Antarctic1.trim.unique.good.filter.fasta as input file for the fasta parameter.
Using Antarctic1.trim.good.names as input file for the name parameter.
1000 984
2000 1976
3000 2962
4000 3941
5000 4917
6000 5896
7000 6880
8000 7864
9000 8856
10000 9838
11000 10815
12000 11797
13000 12776
14000 13753
15000 14742
16000 15728
17000 16708
18000 17680
19000 18657
20000 19635
21000 20612
22000 21589
23000 22566
24000 23545
25000 24517
26000 25496
27000 26473
28000 27455
29000 28419
30000 29396
31000 30368
32000 31344
33000 32317
34000 33297
35000 34277
36000 35257
37000 36233
38000 37205
39000 38182
40000 39163
41000 40146
42000 41123
43000 42100
44000 43083
45000 44053
46000 45032
47000 46017
48000 46985
49000 47960
50000 48931
51000 49902
52000 50881
53000 51861
54000 52836
55000 53814
56000 54794
57000 55772
58000 56752
59000 57735
60000 58716
61000 59691
62000 60676
63000 61655
64000 62634
65000 63596
66000 64570
67000 65549
68000 66515
69000 67491
70000 68463
71000 69437
72000 70413
73000 71381
74000 72365
75000 73341
76000 74320
77000 75301
78000 76280
79000 77256
80000 78227
81000 79207
82000 80181
83000 81156
84000 82133
85000 83103
86000 84076
87000 85049
88000 86023
89000 86998
90000 87960
91000 88937
92000 89906
93000 90885
94000 91861
95000 92838
96000 93806
97000 94789
98000 95756
99000 96728
100000 97710
101000 98675
102000 99658
103000 100631
104000 101603
105000 102580
106000 103553
107000 104522
108000 105503
109000 106482
110000 107461
111000 108441
112000 109414
113000 110393
114000 111363
115000 112338
116000 113313
117000 114284
118000 115262
119000 116242
120000 117231
120568 117783
Output File Names:
Antarctic1.trim.unique.good.filter.names
Antarctic1.trim.unique.good.filter.unique.fasta
mothur > summary.seqs()
Using Antarctic1.trim.unique.good.filter.unique.fasta as input file for the fasta parameter.
Using 2 processors.
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is Antarctic1.trim.unique.good.filter.names which seems to match Antarctic1.trim.unique.good.filter.unique.fasta.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 1326 414 0 3 1
2.5%-tile: 1 1326 467 0 3 2945
25%-tile: 1 1326 477 0 4 29446
Median: 1 1326 480 0 5 58892
75%-tile: 1 1326 482 0 5 88338
97.5%-tile: 1 1326 504 0 7 114839
Maximum: 2 1326 560 0 8 117783
Mean: 1.00002 1326 480.652 0 4.56929
of Seqs: 117783
Output File Names:
Antarctic1.trim.unique.good.filter.unique.summary
mothur > get.current()
Current files saved by mothur: fasta=Antarctic1.trim.unique.good.filter.unique.fasta group=mergegroupsgood name=Antarctic1.trim.unique.good.filter.names processors=2
mothur > pre.cluster(fasta=Antarctic1.trim.unique.good.filter.unique.fasta, name=Antarctic1.trim.unique.good.filter.nmaes, group=mergegroupsgood, diffs=2)
Unable to open Antarctic1.trim.unique.good.filter.nmaes
Using 2 processors.
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is Antarctic1.trim.unique.good.filter.names which seems to match Antarctic1.trim.unique.good.filter.unique.fasta.
[ERROR]: did not complete pre.cluster.
mothur > pre.cluster(fasta=Antarctic1.trim.unique.good.filter.unique.fasta, name=Antarctic1.trim.unique.good.filter.names, group=mergegroupsgood, diffs=2)
Using 2 processors. missing name m130215_173950_42157_c100458282550000001523065103201350_s1_p0/10022 missing name m130215_173950_42157_c100458282550000001523065103201350_s1_p0/10101
etc etc for many sequences…
[ERROR]: Your name file contains 182091 valid sequences, and your groupfile contains 191161, please correct.
/******************************************/
Running command: unique.seqs(fasta=Antarctic1.trim.unique.good.filter.unique.precluster.fasta, name=Antarctic1.trim.unique.good.filter.unique.precluster.names)
[ERROR]: Antarctic1.trim.unique.good.filter.unique.precluster.fasta is blank, aborting.
Using Antarctic1.trim.unique.good.filter.unique.fasta as input file for the fasta parameter.
[ERROR]: Antarctic1.trim.unique.good.filter.unique.precluster.names is blank, aborting.
/******************************************/
Segmentation fault