pre.cluster bugs

mothur bugs
1

Hi,
I came across with the problem in pre.cluster(). This problem troubles me for several days.
Now I use the lastest version 1.39.5.
The precluseter problem always pops up like this:

Total number of sequences before pre.cluster was 8589.
pre.cluster removed 5202 sequences.

It took 3 secs to cluster 8589 sequences.
[ERROR]: group M02171_86_000000000-D0H8A_1_1101_16960_1442,M02171_86_000000000-D0H8A_1_1101_8508_3557,M02171_86_000000000-D0H8A_1_1101_8560_3363,M02171_86_000000000-D0H8A_1_1101_10986_2455,M02171_86_000000000-D0H8A_1_1101_8616_13508,M02171_86_000000000-D0H8A_1_1101_16497_1715,M02171_86_000000000-D0H8A_1_1101_10082_2818,M02171_86_000000000-D0H8A_1_1101_9827_2981,M02171_86_000000000-D0H8A_1_1101_13949_2169,M02171_86_000000000-D0H8A_1_1101_14082_6431,M02171_86_000000000-D0H8A_1_1101_10999_3842,M02171_86_000000000-D0H8A_1_1102_22666_9673,M02171_86_000000000-D0H8A_1_1101_12769_5875,M02171_86_000000000-D0H8A_1_1101_14889_12800,M02171_86_000000000-D0H8A_1_1101_17131_13837,M02171_86_000000000-D0H8A_1_1101_11658_8559,M02171_86_000000000-D0H8A_1_1101_11568_4117,M02171_86_000000000-D0H8A_1_1101_3275_18157,M02171_86_000000000-D0H8A_1_1101_10980_2335,M02171_86_000000000-D0H8A_1_1101_17533_20452,M02171_86_000000000-D0H8A_1_1101_7206_10806,M02171_86_000000000-D0H8A_1_1101_19836_26119,M02171_86_000000000-D0H8A_1_1101_5280_8952,M02171_86_000000000-D0H8A_1_1101_14519_9212,M02171_86_000000000-D0H8A_1_1101_8513_10047,M02171_86_000000000-D0H8A_1_1102_25473_18812,M02171_86_000000000-D0H8A_1_1101_9146_9431,M02171_86_000000000-D0H8A_1_1101_7542_9568,M02171_86_000000000-D0H8A_1_1101_25655_19355,M02171_86_000000000-D0H8A_1_1101_4646_19229,M02171_86_000000000-D0H8A_1_1101_10122_19655,M02171_86_000000000-D0H8A_1_1101_14731_17974,M02171_86_000000000-D0H8A_1_1101_15488_12623,M02171_86_000000000-D0H8A_1_1102_24837_4714,M02171_86_000000000-D0H8A_1_1101_6574_12051,M02171_86_000000000-D0H8A_1_1102_27110_13874,M02171_86_000000000-D0H8A_1_1101_11200_14842,M02171_86_000000000-D0H8A_1_1102_18918_13986,M02171_86_000000000-D0H8A_1_1101_19017_22269,M02171_86_000000000-D0H8A_1_1101_22728_13412,M02171_86_000000000-D0H8A_1_1101_13672_6126,M02171_86_000000000-D0H8A_1_1101_8960_7806,M02171_86_000000000-D0H8A_1_1101_16638_3702,M02171_86_000000000-D0H8A_1_1101_20161_7714,M02171_86_000000000-D0H8A_1_1101_21652_3985,M02171_86_000000000-D0H8A_1_1101_9028_8039,M02171_86_000000000-D0H8A_1_1101_16840_5553,M02171_86_000000000-D0H8A_1_1102_5315_7615,M02171_86_000000000-D0H8A_1_1102_26148_7769,M02171_86_000000000-D0H8A_1_1102_4152_7718,M02171_86_000000000-D0H8A_1_1102_26997_11249,M02171_86_000000000-D0H8A_1_1102_19208_25141,M02171_86_000000000-D0H8A_1_1102_6974_25644,M02171_86_000000000-D0H8A_1_1102_11588_10135,M02171_86_000000000-D0H8A_1_1102_21366_9676,M02171_86_000000000-D0H8A_1_1102_25472_9624,M02171_86_000000000-D0H8A_1_1102_23981_9845,M02171_86_000000000-D0H8A_1_1102_11275_10849,M02171_86_000000000-D0H8A_1_1102_15652_2528,M02171_86_000000000-D0H8A_1_1102_20313_2294,M02171_86_000000000-D0H8A_1_1102_16554_3917,M02171_86_000000000-D0H8A_1_1102_19163_3415,M02171_86_000000000-D0H8A_1_1102_14759_1642,M02171_86_000000000-D0H8A_1_1102_17372_6199,M02171_86_000000000-D0H8A_1_1102_25828_5852,M02171_86_000000000-D0H8A_1_1102_13924_5732,M02171_86_000000000-D0H8A_1_1102_14651_5337,M02171_86_000000000-D0H8A_1_1102_11467_5335,M02171_86_000000000-D0H8A_1_1102_23939_5231,M02171_86_000000000-D0H8A_1_1102_7078_5548,M02171_86_000000000-D0H8A_1_1102_9507_21887,M02171_86_000000000-D0H8A_1_1102_17392_22404,M02171_86_000000000-D0H8A_1_1102_19110_22289,M02171_86_000000000-D0H8A_1_1102_4953_20882,M02171_86_000000000-D0H8A_1_1102_26052_20982,M02171_86_000000000-D0H8A_1_1102_26430_19567,M02171_86_000000000-D0H8A_1_1102_24770_20643,M02171_86_000000000-D0H8A_1_1102_18394_20662,M02171_86_000000000-D0H8A_1_1102_7336_20256,M02171_86_000000000-D0H8A_1_1102_18536_21376,M02171_86_000000000-D0H8A_1_1102_17205_19166,M02171_86_000000000-D0H8A_1_1102_18852_14255,M02171_86_000000000-D0H8A_1_1102_5249_14041,M02171_86_000000000-D0H8A_1_1102_5384_13979,M02171_86_000000000-D0H8A_1_1102_10973_12476,M02171_86_000000000-D0H8A_1_1102_19313_13144,M02171_86_000000000-D0H8A_1_1102_9993_12835,M02171_86_000000000-D0H8A_1_1102_20372_22935,M02171_86_000000000-D0H8A_1_1102_8608_23211,M02171_86_000000000-D0H8A_1_1102_19364_22818,M02171_86_000000000-D0H8A_1_1102_19508_14530,M02171_86_000000000-D0H8A_1_1102_18131_14700,M02171_86_000000000-D0H8A_1_1101_7399_10268,M02171_86_000000000-D0H8A_1_1101_17481_10167,M02171_86_000000000-D0H8A_1_1101_19727_9752,M02171_86_000000000-D0H8A_1_1101_21684_9743,M02171_86_000000000-D0H8A_1_1101_13731_10740,M02171_86_000000000-D0H8A_1_1101_13720_10723,M02171_86_000000000-D0H8A_1_1101_24270_10688,M02171_86_000000000-D0H8A_1_1101_17895_10597,M02171_86_000000000-D0H8A_1_1101_12111_8694,M02171_86_000000000-D0H8A_1_1101_27548_8817,M02171_86_000000000-D0H8A_1_1101_11525_8798,M02171_86_000000000-D0H8A_1_1101_23645_8489,M02171_86_000000000-D0H8A_1_1101_25678_8431,M02171_86_000000000-D0H8A_1_1101_14219_9251,M02171_86_000000000-D0H8A_1_1101_9908_9230,M02171_86_000000000-D0H8A_1_1101_10532_9073,M02171_86_000000000-D0H8A_1_1101_16542_13147,M02171_86_000000000-D0H8A_1_1101_2967_13181,M02171_86_000000000-D0H8A_1_1101_20658_12727,M02171_86_000000000-D0H8A_1_1101_11362_12788,M02171_86_000000000-D0H8A_1_1101_28431_13856,M02171_86_000000000-D0H8A_1_1101_17421_11498,M02171_86_000000000-D0H8A_1_1101_18452_11715,M02171_86_000000000-D0H8A_1_1101_17439_11608,M02171_86_000000000-D0H8A_1_1101_16928_11337,M02171_86_000000000-D0H8A_1_1101_21329_12428,M02171_86_000000000-D0H8A_1_1101_13930_12543,M02171_86_000000000-D0H8A_1_1101_27566_11847,M02171_86_000000000-D0H8A_1_1101_14395_4079,M02171_86_000000000-D0H8A_1_1101_13057_3936,M02171_86_000000000-D0H8A_1_1101_25000_4999,M02171_86_000000000-D0H8A_1_1101_24902_4974,M02171_86_000000000-D0H8A_1_1101_11560_4640,M02171_86_000000000-D0H8A_1_1101_24296_4853,M02171_86_000000000-D0H8A_1_1101_20582_2261,M02171_86_000000000-D0H8A_1_1101_12798_2158,M02171_86_000000000-D0H8A_1_1101_20915_3018,M02171_86_000000000-D0H8A_1_1101_12704_2851,M02171_86_000000000-D0H8A_1_1101_14666_7265,M02171_86_000000000-D0H8A_1_1101_7276_6875,M02171_86_000000000-D0H8A_1_1101_13897_6943,M02171_86_000000000-D0H8A_1_1101_21042_7045,M02171_86_000000000-D0H8A_1_1101_24748_7876,M02171_86_000000000-D0H8A_1_1101_14235_7695,M02171_86_000000000-D0H8A_1_1101_25184_7507,M02171_86_000000000-D0H8A_1_1101_6199_6017,M02171_86_000000000-D0H8A_1_1101_11418_5954,M02171_86_000000000-D0H8A_1_1101_19197_6133,M02171_86_000000000-D0H8A_1_1101_13698_6420,M02171_86_000000000-D0H8A_1_1101_12872_6355,M02171_86_000000000-D0H8A_1_1101_17150_6328,M02171_86_000000000-D0H8A_1_1101_8060_21871,M02171_86_000000000-D0H8A_1_1101_18810_22169,M02171_86_000000000-D0H8A_1_1101_19813_21723,M02171_86_000000000-D0H8A_1_1101_6121_21662,M02171_86_000000000-D0H8A_1_1101_25607_20313,M02171_86_000000000-D0H8A_1_1101_8253_20116,M02171_86_000000000-D0H8A_1_1101_18153_20022,M02171_86_000000000-D0H8A_1_1101_19656_21342,M02171_86_000000000-D0H8A_1_1101_16925_21049,M02171_86_000000000-D0H8A_1_1101_21681_25889,M02171_86_000000000-D0H8A_1_1101_15414_25261,M02171_86_000000000-D0H8A_1_1101_13521_25436,M02171_86_000000000-D0H8A_1_1101_18564_26968,M02171_86_000000000-D0H8A_1_1101_22906_26220,M02171_86_000000000-D0H8A_1_1101_17480_26111,M02171_86_000000000-D0H8A_1_1101_5258_23403,M02171_86_000000000-D0H8A_1_1101_2432_15600,M02171_86_000000000-D0H8A_1_1101_2440_15584,M02171_86_000000000-D0H8A_1_1101_21188_15692,M02171_86_000000000-D0H8A_1_1101_25981_16311,M02171_86_000000000-D0H8A_1_1101_21959_14491,M02171_86_000000000-D0H8A_1_1101_2793_14173,M02171_86_000000000-D0H8A_1_1101_24716_14378,M02171_86_000000000-D0H8A_1_1101_9901_18990,M02171_86_000000000-D0H8A_1_1101_21872_19022,M02171_86_000000000-D0H8A_1_1101_13820_18511,M02171_86_000000000-D0H8A_1_1101_3109_18456,M02171_86_000000000-D0H8A_1_1101_10498_19920,M02171_86_000000000-D0H8A_1_1101_27923_19897,M02171_86_000000000-D0H8A_1_1101_28912_19477,M02171_86_000000000-D0H8A_1_1101_26731_17219,M02171_86_000000000-D0H8A_1_1101_11983_18175,M02171_86_000000000-D0H8A_1_1101_18234_18047 is not in your count table. Please correct.

However, when I ran this data for 3 times. Two times caused the error. The last time I got the correct result. Then I ran another batch of data. The same problem pops up. Then I ran it also for another time, the porblem was solved.

I can not figure out what is the problem. Then I think it over and over, then I change to install an older version of mothur 1.37.6. Everything is ok now.

I there anyone who can fix this problem? Thanks in advance.

Enclosed is one of my code and run procedure.

mothur > make.contigs(ffastq=Miseq2nd_R1.fastq,rfastq=Miseq2nd_R2.fastq,findex=Miseq2nd_I.fastq,oligos=Oligo_II.txt,processors=16)

Using 16 processors.
Making contigs...
1000
...
56720
Done.
It took 59 secs to process 905989 sequences.

Group count: 
Acetate 35767
Alanine 35713
Benzonate 64588
Butyrate 2771
Ethanol 36024
Formate 9276
Lactate 43848
Propionate 32753
Succinate 50653

Total of all groups is 311393

Output File Names: 
Miseq2nd_R1.trim.contigs.fasta
Miseq2nd_R1.scrap.contigs.fasta
Miseq2nd_R1.trim.contigs.qual
Miseq2nd_R1.scrap.contigs.qual
Miseq2nd_R1.contigs.report
Miseq2nd_R1.contigs.groups

[WARNING]: your sequence names contained ':'.  I changed them to '_' to avoid problems in your downstream analysis.

mothur > summary.seqs(fasta=Miseq2nd_R1.trim.contigs.fasta)

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 152 152 0 3 1
2.5%-tile: 1 252 252 0 4 7785
25%-tile: 1 253 253 0 5 77849
Median:  1 253 253 0 5 155697
75%-tile: 1 254 254 0 5 233545
97.5%-tile: 1 254 254 1 6 303609
Maximum: 1 302 302 49 147 311393
Mean: 1 253.433 253.433 0.137485 4.92596
# of Seqs: 311393

Output File Names: 
Miseq2nd_R1.trim.contigs.summary

It took 1 secs to summarize 311393 sequences.

mothur > screen.seqs(fasta=Miseq2nd_R1.trim.contigs.fasta, group=Miseq2nd_R1.contigs.groups, summary=Miseq2nd_R1.trim.contigs.summary, maxambig=0, maxlength=254)

Using 16 processors.
...
Processing sequence: 19496

Output File Names: 
Miseq2nd_R1.trim.contigs.good.summary
Miseq2nd_R1.trim.contigs.good.fasta
Miseq2nd_R1.trim.contigs.bad.accnos
Miseq2nd_R1.contigs.good.groups


It took 9 secs to screen 311393 sequences.

mothur > summary.seqs(fasta=Miseq2nd_R1.trim.contigs.good.fasta)

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 152 152 0 3 1
2.5%-tile: 1 252 252 0 4 7328
25%-tile: 1 253 253 0 5 73278
Median:  1 253 253 0 5 146556
75%-tile: 1 254 254 0 5 219834
97.5%-tile: 1 254 254 0 6 285784
Maximum: 1 254 254 0 73 293111
Mean: 1 253.257 253.257 0 4.90673
# of Seqs: 293111

Output File Names: 
Miseq2nd_R1.trim.contigs.good.summary

It took 1 secs to summarize 293111 sequences.

mothur > unique.seqs(fasta=Miseq2nd_R1.trim.contigs.good.fasta)
1000 381
...
293111 38824

Output File Names: 
Miseq2nd_R1.trim.contigs.good.names
Miseq2nd_R1.trim.contigs.good.unique.fasta


mothur > count.seqs(name=Miseq2nd_R1.trim.contigs.good.names, group=Miseq2nd_R1.contigs.good.groups)

Using 16 processors.
It took 2 secs to create a table for 293111 sequences.


Total number of sequences: 293111

Output File Names: 
Miseq2nd_R1.trim.contigs.good.count_table


mothur > summary.seqs(count=Miseq2nd_R1.trim.contigs.good.count_table)
Using Miseq2nd_R1.trim.contigs.good.unique.fasta as input file for the fasta parameter.

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 152 152 0 3 1
2.5%-tile: 1 252 252 0 4 7328
25%-tile: 1 253 253 0 5 73278
Median:  1 253 253 0 5 146556
75%-tile: 1 254 254 0 5 219834
97.5%-tile: 1 254 254 0 6 285784
Maximum: 1 254 254 0 73 293111
Mean: 1 253.257 253.257 0 4.90673
# of unique seqs: 38824
total # of seqs: 293111

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.summary

It took 0 secs to summarize 293111 sequences.

mothur > align.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.fasta, reference=silva.bacteria.pcr.fasta)

Using 16 processors.

Reading in the silva.bacteria.pcr.fasta template sequences... DONE.
It took 6 to read  14956 sequences.
Aligning sequences from Miseq2nd_R1.trim.contigs.good.unique.fasta ...
100
...
2429
[WARNING]: Some of your sequences generated alignments that eliminated too many bases, a list is provided in Miseq2nd_R1.trim.contigs.good.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well.
It took 11 secs to align 38824 sequences.


Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.align
Miseq2nd_R1.trim.contigs.good.unique.align.report
Miseq2nd_R1.trim.contigs.good.unique.flip.accnos


mothur > summary.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.align, count=Miseq2nd_R1.trim.contigs.good.count_table)

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 1968 11550 252 0 4 7328
25%-tile: 1968 11550 253 0 5 73278
Median:  1968 11550 253 0 5 146556
75%-tile: 1968 11550 254 0 5 219834
97.5%-tile: 1968 11550 254 0 6 285784
Maximum: 13425 13425 254 0 73 293111
Mean: 1971.75 11548.7 253.153 0 4.90489
# of unique seqs: 38824
total # of seqs: 293111

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.summary

It took 1 secs to summarize 293111 sequences.

mothur > screen.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.align, count=Miseq2nd_R1.trim.contigs.good.count_table, summary=Miseq2nd_R1.trim.contigs.good.unique.summary, start=1968, end=11550, maxhomop=8)

Using 16 processors.
Processing sequence: 100
...
Processing sequence: 2426

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.good.summary
Miseq2nd_R1.trim.contigs.good.unique.good.align
Miseq2nd_R1.trim.contigs.good.unique.bad.accnos
Miseq2nd_R1.trim.contigs.good.good.count_table


It took 6 secs to screen 38824 sequences.

mothur > summary.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.good.align, count=Miseq2nd_R1.trim.contigs.good.good.count_table)

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1967 11550 245 0 3 1
2.5%-tile: 1968 11550 252 0 4 7316
25%-tile: 1968 11550 253 0 5 73156
Median:  1968 11550 253 0 5 146312
75%-tile: 1968 11550 254 0 5 219468
97.5%-tile: 1968 11550 254 0 6 285308
Maximum: 1968 11553 254 0 8 292623
Mean: 1968 11550 253.289 0 4.90181
# of unique seqs: 38357
total # of seqs: 292623

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.good.summary

It took 1 secs to summarize 292623 sequences.

mothur > filter.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.good.align, vertical=T, trump=.)

Using 16 processors.
Creating Filter... 
100
...
2397

Length of filtered alignment: 441
Number of columns removed: 12984
Length of the original alignment: 13425
Number of sequences used to construct filter: 38357

Output File Names: 
Miseq2nd_R1.filter
Miseq2nd_R1.trim.contigs.good.unique.good.filter.fasta


mothur > unique.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.good.filter.fasta, count=Miseq2nd_R1.trim.contigs.good.good.count_table)
1000 1000
...
38357 38357

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.good.filter.count_table
Miseq2nd_R1.trim.contigs.good.unique.good.filter.unique.fasta


mothur > summary.seqs(fasta=Miseq2nd_R1.trim.contigs.good.unique.good.filter.unique.fasta,count=Miseq2nd_R1.trim.contigs.good.unique.good.filter.count_table)

Using 16 processors.

  Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 440 245 0 3 1
2.5%-tile: 1 441 252 0 4 7316
25%-tile: 1 441 253 0 5 73156
Median:  1 441 253 0 5 146312
75%-tile: 1 441 254 0 5 219468
97.5%-tile: 1 441 254 0 6 285308
Maximum: 1 441 254 0 8 292623
Mean: 1 441 253.289 0 4.90181
# of unique seqs: 38357
total # of seqs: 292623

Output File Names: 
Miseq2nd_R1.trim.contigs.good.unique.good.filter.unique.summary

It took 0 secs to summarize 292623 sequences.

mothur > pre.cluster(fasta=Miseq2nd_R1.trim.contigs.good.unique.good.filter.unique.fasta, count=Miseq2nd_R1.trim.contigs.good.unique.good.filter.count_table, diffs=2)

Using 16 processors.

Processing group Ethanol:

Processing group Benzonate:

Processing group Lactate:

Processing group Succinate:

Processing group Butyrate:

Processing group Formate:
0 455 63
100 246 272

Processing group Alanine:
200 244 274
0 1003 184
300 234 284
400 229 289
500 226 292
518 226 292
Total number of sequences before pre.cluster was 518.
pre.cluster removed 292 sequences.


Processing group Propionate:
100 522 665

Processing group Acetate:
It took 0 secs to cluster 518 sequences.
200 507 680
300 487 700
400 475 712
0 5187 271
500 473 714
600 468 719
700 464 723
800 462 725
900 458 729
1000 455 732
0 4390 412
0 5377 716
1100 455 732
1187 455 732
Total number of sequences before pre.cluster was 1187.
pre.cluster removed 732 sequences.

0 6763 486
0 4107 581
It took 0 secs to cluster 1187 sequences.
0 7269 1320
0 6891 175
100 1924 2878
100 2545 2913
100 2835 3258
200 1864 2938
100 2212 2476
300 1834 2968
200 2713 3380
200 2352 3106
400 1809 2993
100 3802 3447
500 1771 3031
300 2309 3149
200 2077 2611
300 2662 3431
600 1756 3046
100 4473 4116
400 2639 3454
400 2277 3181
700 1749 3053
300 2020 2668
800 1720 3082
500 2256 3202
200 3619 3630
900 1709 3093
400 2001 2687
500 2584 3509
600 2248 3210
600 2578 3515
1000 1694 3108
500 1989 2699
700 2219 3239
700 2553 3540
1100 1680 3122
200 4224 4365
600 1975 2713
800 2204 3254
800 2532 3561
300 3452 3797
700 1961 2727
100 4391 2675
1200 1675 3127
900 2179 3279
900 2508 3585
300 4144 4445
800 1943 2745
1000 2168 3290
1000 2496 3597
1300 1661 3141
400 3362 3887
900 1936 2752
1100 2151 3307
1100 2487 3606
1000 1928 2760
1400 1649 3153
400 4086 4503
500 3337 3912
1200 2471 3622
1500 1648 3154
1200 2142 3316
1100 1910 2778
1300 2461 3632
1600 1643 3159
500 4042 4547
1400 2450 3643
600 3304 3945
1700 1638 3164
1300 2135 3323
1200 1904 2784
1800 1632 3170
600 4006 4583
700 3267 3982
200 4005 3061
1900 1629 3173
1400 2126 3332
1500 2427 3666
1300 1895 2793
700 3950 4639
2000 1623 3179
800 3241 4008
800 3879 4710
2100 1618 3184
1500 2124 3334
1400 1885 2803
2200 1614 3188
1600 2418 3675
900 3226 4023
1500 1881 2807
1600 2122 3336
900 3857 4732
2300 1611 3191
2400 1611 3191
2500 1608 3194
1000 3839 4750
1000 3206 4043
1600 1876 2812
1700 2108 3350
1700 2405 3688
2600 1607 3195
300 3790 3276
1100 3816 4773
2700 1602 3200
1100 3178 4071
1700 1866 2822
1800 2101 3357
1200 3783 4806
2800 1602 3200
2900 1601 3201
1800 1860 2828
1800 2393 3700
1200 3163 4086
3000 1597 3205
1300 3778 4811
1900 2094 3364
3100 1595 3207
1300 3154 4095
3200 1592 3210
1900 1854 2834
1400 3755 4834
3300 1588 3214
3400 1587 3215
1400 3143 4106
3500 1587 3215
2000 2087 3371
1900 2378 3715
3600 1586 3216
1500 3751 4838
3700 1584 3218
2000 1852 2836
3800 1584 3218
3900 1583 3219
400 3636 3430
4000 1583 3219
2100 2083 3375
4100 1583 3219
2000 2361 3732
4200 1582 3220
4300 1581 3221
1600 3744 4845
2100 1848 2840
4400 1581 3221
4500 1580 3222
4600 1579 3223
4700 1578 3224
4800 1578 3224
4802 1578 3224
Total number of sequences before pre.cluster was 4802.
pre.cluster removed 3224 sequences.

1500 3133 4116
2200 2082 3376
1700 3731 4858
2200 1842 2846
2100 2350 3743
2300 2072 3386
2300 1837 2851
2400 2070 3388
1600 3130 4119
2400 1832 2856
2200 2344 3749
It took 0 secs to cluster 4802 sequences.
500 3556 3510
2500 2068 3390
2500 1823 2865
1800 3720 4869
2300 2340 3753
2600 1821 2867
2600 2063 3395
1700 3118 4131
2700 1819 2869
2700 2062 3396
2800 1819 2869
2400 2333 3760
2900 1816 2872
2800 2057 3401
600 3489 3577
3000 1815 2873
3100 1813 2875
2900 2053 3405
2500 2330 3763
3200 1808 2880
1900 3710 4879
3300 1803 2885
1800 3102 4147
3000 2051 3407
3400 1800 2888
3500 1799 2889
3600 1797 2891
3700 1796 2892
3100 2051 3407
2600 2325 3768
3800 1795 2893
700 3449 3617
3900 1793 2895
3200 2043 3415
4000 1793 2895
4100 1793 2895
4200 1792 2896
4300 1791 2897
3300 2037 3421
4400 1788 2900
2700 2322 3771
4500 1788 2900
4600 1787 2901
4688 1787 2901
Total number of sequences before pre.cluster was 4688.
pre.cluster removed 2901 sequences.

1900 3086 4163
2000 3701 4888
3400 2030 3428
3500 2028 3430
2800 2314 3779
800 3418 3648
3600 2026 3432
3700 2025 3433
2000 3083 4166
2900 2307 3786
3800 2022 3436
3900 2020 3438
It took 1 secs to cluster 4688 sequences.
4000 2016 3442
2100 3659 4930
3000 2306 3787
4100 2014 3444
4200 2013 3445
900 3383 3683
2100 3076 4173
4300 2012 3446
4400 2009 3449
3100 2298 3795
4500 2007 3451
4600 2007 3451
4700 2007 3451
4800 2006 3452
4900 2006 3452
3200 2294 3799
5000 2005 3453
2200 3063 4186
5100 2004 3454
1000 3358 3708
2200 3650 4939
5200 2002 3456
5300 2002 3456
5400 2001 3457
5458 2001 3457
Total number of sequences before pre.cluster was 5458.
pre.cluster removed 3457 sequences.

3300 2286 3807
3400 2278 3815
2300 3055 4194
1100 3344 3722
3500 2269 3824
2300 3635 4954
3600 2266 3827
2400 3047 4202
It took 1 secs to cluster 5458 sequences.
1200 3326 3740
3700 2264 3829
3800 2261 3832
2500 3042 4207
3900 2258 3835
4000 2248 3845
1300 3308 3758
2400 3629 4960
4100 2246 3847
4200 2244 3849
2600 3036 4213
4300 2240 3853
1400 3287 3779
4400 2238 3855
4500 2235 3858
4600 2234 3859
2700 3025 4224
4700 2233 3860
4800 2233 3860
2500 3612 4977
4900 2232 3861
1500 3273 3793
5000 2230 3863
5100 2229 3864
5200 2229 3864
5300 2229 3864
2800 3017 4232
5400 2228 3865
5500 2228 3865
1600 3255 3811
5600 2228 3865
5700 2226 3867
5800 2226 3867
5900 2225 3868
6000 2224 3869
6093 2224 3869
Total number of sequences before pre.cluster was 6093.
pre.cluster removed 3869 sequences.

2600 3606 4983
2900 3014 4235
1700 3252 3814
3000 3011 4238
2700 3597 4992
1800 3236 3830
It took 1 secs to cluster 6093 sequences.
3100 3009 4240
1900 3233 3833
3200 3007 4242
2800 3591 4998
3300 3004 4245
2000 3229 3837
2900 3586 5003
2100 3227 3839
3400 3003 4246
3500 2999 4250
2200 3221 3845
3000 3578 5011
3600 2988 4261
2300 3219 3847
3700 2987 4262
3100 3571 5018
2400 3211 3855
3800 2983 4266
2500 3206 3860
3900 2980 4269
2600 3206 3860
3200 3558 5031
4000 2978 4271
2700 3199 3867
4100 2975 4274
3300 3555 5034
4200 2974 4275
2800 3195 3871
4300 2969 4280
3400 3554 5035
2900 3189 3877
4400 2966 4283
4500 2962 4287
3000 3182 3884
3500 3535 5054
4600 2958 4291
3100 3180 3886
4700 2958 4291
4800 2957 4292
3200 3174 3892
4900 2953 4296
3600 3522 5067
5000 2952 4297
5100 2950 4299
3300 3170 3896
5200 2949 4300
5300 2947 4302
3400 3164 3902
3700 3514 5075
5400 2946 4303
5500 2944 4305
3500 3160 3906
5600 2944 4305
5700 2942 4307
3800 3506 5083
5800 2942 4307
3600 3159 3907
5900 2939 4310
6000 2934 4315
3700 3156 3910
6100 2932 4317
6200 2932 4317
3900 3502 5087
6300 2930 4319
3800 3150 3916
6400 2929 4320
6500 2928 4321
6600 2927 4322
6700 2926 4323
3900 3145 3921
6800 2926 4323
6900 2925 4324
7000 2923 4326
4000 3490 5099
7100 2922 4327
7200 2922 4327
7249 2922 4327
Total number of sequences before pre.cluster was 7249.
pre.cluster removed 4327 sequences.

4000 3140 3926
4100 3490 5099
4100 3140 3926
4200 3138 3928
4200 3487 5102
4300 3133 3933
4400 3123 3943
It took 2 secs to cluster 7249 sequences.
4500 3122 3944
4300 3480 5109
4600 3121 3945
4700 3119 3947
4400 3478 5111
4800 3117 3949
4900 3115 3951
4500 3472 5117
5000 3115 3951
5100 3114 3952
5200 3113 3953
4600 3463 5126
5300 3112 3954
4700 3460 5129
5400 3111 3955
5500 3110 3956
5600 3108 3958
4800 3458 5131
5700 3105 3961
5800 3101 3965
5900 3098 3968
6000 3097 3969
4900 3456 5133
6100 3097 3969
6200 3096 3970
6300 3096 3970
6400 3094 3972
5000 3453 5136
6500 3093 3973
6600 3092 3974
6700 3090 3976
6800 3090 3976
6900 3089 3977
7000 3087 3979
7066 3084 3982
Total number of sequences before pre.cluster was 7066.
pre.cluster removed 3982 sequences.

5100 3446 5143
5200 3444 5145
5300 3439 5150
5400 3435 5154
5500 3434 5155
5600 3433 5156
5700 3430 5159
It took 2 secs to cluster 7066 sequences.
5800 3427 5162
5900 3426 5163
6000 3424 5165
6100 3420 5169
6200 3418 5171
6300 3415 5174
6400 3414 5175
6500 3412 5177
6600 3408 5181
6700 3406 5183
6800 3405 5184
6900 3404 5185
7000 3403 5186
7100 3400 5189
7200 3398 5191
7300 3398 5191
7400 3396 5193
7500 3395 5194
7600 3393 5196
7700 3391 5198
7800 3389 5200
7900 3389 5200
8000 3389 5200
8100 3389 5200
8200 3389 5200
8300 3389 5200
8400 3389 5200
8500 3388 5201
8589 3387 5202
Total number of sequences before pre.cluster was 8589.
pre.cluster removed 5202 sequences.

It took 3 secs to cluster 8589 sequences.
[ERROR]: group M02171_86_000000000-D0H8A_1_1101_16960_1442,M02171_86_000000000-D0H8A_1_1101_8508_3557,M02171_86_000000000-D0H8A_1_1101_8560_3363,M02171_86_000000000-D0H8A_1_1101_10986_2455,M02171_86_000000000-D0H8A_1_1101_8616_13508,M02171_86_000000000-D0H8A_1_1101_16497_1715,M02171_86_000000000-D0H8A_1_1101_10082_2818,M02171_86_000000000-D0H8A_1_1101_9827_2981,M02171_86_000000000-D0H8A_1_1101_13949_2169,M02171_86_000000000-D0H8A_1_1101_14082_6431,M02171_86_000000000-D0H8A_1_1101_10999_3842,M02171_86_000000000-D0H8A_1_1102_22666_9673,M02171_86_000000000-D0H8A_1_1101_12769_5875,M02171_86_000000000-D0H8A_1_1101_14889_12800,M02171_86_000000000-D0H8A_1_1101_17131_13837,M02171_86_000000000-D0H8A_1_1101_11658_8559,M02171_86_000000000-D0H8A_1_1101_11568_4117,M02171_86_000000000-D0H8A_1_1101_3275_18157,M02171_86_000000000-D0H8A_1_1101_10980_2335,M02171_86_000000000-D0H8A_1_1101_17533_20452,M02171_86_000000000-D0H8A_1_1101_7206_10806,M02171_86_000000000-D0H8A_1_1101_19836_26119,M02171_86_000000000-D0H8A_1_1101_5280_8952,M02171_86_000000000-D0H8A_1_1101_14519_9212,M02171_86_000000000-D0H8A_1_1101_8513_10047,M02171_86_000000000-D0H8A_1_1102_25473_18812,M02171_86_000000000-D0H8A_1_1101_9146_9431,M02171_86_000000000-D0H8A_1_1101_7542_9568,M02171_86_000000000-D0H8A_1_1101_25655_19355,M02171_86_000000000-D0H8A_1_1101_4646_19229,M02171_86_000000000-D0H8A_1_1101_10122_19655,M02171_86_000000000-D0H8A_1_1101_14731_17974,M02171_86_000000000-D0H8A_1_1101_15488_12623,M02171_86_000000000-D0H8A_1_1102_24837_4714,M02171_86_000000000-D0H8A_1_1101_6574_12051,M02171_86_000000000-D0H8A_1_1102_27110_13874,M02171_86_000000000-D0H8A_1_1101_11200_14842,M02171_86_000000000-D0H8A_1_1102_18918_13986,M02171_86_000000000-D0H8A_1_1101_19017_22269,M02171_86_000000000-D0H8A_1_1101_22728_13412,M02171_86_000000000-D0H8A_1_1101_13672_6126,M02171_86_000000000-D0H8A_1_1101_8960_7806,M02171_86_000000000-D0H8A_1_1101_16638_3702,M02171_86_000000000-D0H8A_1_1101_20161_7714,M02171_86_000000000-D0H8A_1_1101_21652_3985,M02171_86_000000000-D0H8A_1_1101_9028_8039,M02171_86_000000000-D0H8A_1_1101_16840_5553,M02171_86_000000000-D0H8A_1_1102_5315_7615,M02171_86_000000000-D0H8A_1_1102_26148_7769,M02171_86_000000000-D0H8A_1_1102_4152_7718,M02171_86_000000000-D0H8A_1_1102_26997_11249,M02171_86_000000000-D0H8A_1_1102_19208_25141,M02171_86_000000000-D0H8A_1_1102_6974_25644,M02171_86_000000000-D0H8A_1_1102_11588_10135,M02171_86_000000000-D0H8A_1_1102_21366_9676,M02171_86_000000000-D0H8A_1_1102_25472_9624,M02171_86_000000000-D0H8A_1_1102_23981_9845,M02171_86_000000000-D0H8A_1_1102_11275_10849,M02171_86_000000000-D0H8A_1_1102_15652_2528,M02171_86_000000000-D0H8A_1_1102_20313_2294,M02171_86_000000000-D0H8A_1_1102_16554_3917,M02171_86_000000000-D0H8A_1_1102_19163_3415,M02171_86_000000000-D0H8A_1_1102_14759_1642,M02171_86_000000000-D0H8A_1_1102_17372_6199,M02171_86_000000000-D0H8A_1_1102_25828_5852,M02171_86_000000000-D0H8A_1_1102_13924_5732,M02171_86_000000000-D0H8A_1_1102_14651_5337,M02171_86_000000000-D0H8A_1_1102_11467_5335,M02171_86_000000000-D0H8A_1_1102_23939_5231,M02171_86_000000000-D0H8A_1_1102_7078_5548,M02171_86_000000000-D0H8A_1_1102_9507_21887,M02171_86_000000000-D0H8A_1_1102_17392_22404,M02171_86_000000000-D0H8A_1_1102_19110_22289,M02171_86_000000000-D0H8A_1_1102_4953_20882,M02171_86_000000000-D0H8A_1_1102_26052_20982,M02171_86_000000000-D0H8A_1_1102_26430_19567,M02171_86_000000000-D0H8A_1_1102_24770_20643,M02171_86_000000000-D0H8A_1_1102_18394_20662,M02171_86_000000000-D0H8A_1_1102_7336_20256,M02171_86_000000000-D0H8A_1_1102_18536_21376,M02171_86_000000000-D0H8A_1_1102_17205_19166,M02171_86_000000000-D0H8A_1_1102_18852_14255,M02171_86_000000000-D0H8A_1_1102_5249_14041,M02171_86_000000000-D0H8A_1_1102_5384_13979,M02171_86_000000000-D0H8A_1_1102_10973_12476,M02171_86_000000000-D0H8A_1_1102_19313_13144,M02171_86_000000000-D0H8A_1_1102_9993_12835,M02171_86_000000000-D0H8A_1_1102_20372_22935,M02171_86_000000000-D0H8A_1_1102_8608_23211,M02171_86_000000000-D0H8A_1_1102_19364_22818,M02171_86_000000000-D0H8A_1_1102_19508_14530,M02171_86_000000000-D0H8A_1_1102_18131_14700,M02171_86_000000000-D0H8A_1_1101_7399_10268,M02171_86_000000000-D0H8A_1_1101_17481_10167,M02171_86_000000000-D0H8A_1_1101_19727_9752,M02171_86_000000000-D0H8A_1_1101_21684_9743,M02171_86_000000000-D0H8A_1_1101_13731_10740,M02171_86_000000000-D0H8A_1_1101_13720_10723,M02171_86_000000000-D0H8A_1_1101_24270_10688,M02171_86_000000000-D0H8A_1_1101_17895_10597,M02171_86_000000000-D0H8A_1_1101_12111_8694,M02171_86_000000000-D0H8A_1_1101_27548_8817,M02171_86_000000000-D0H8A_1_1101_11525_8798,M02171_86_000000000-D0H8A_1_1101_23645_8489,M02171_86_000000000-D0H8A_1_1101_25678_8431,M02171_86_000000000-D0H8A_1_1101_14219_9251,M02171_86_000000000-D0H8A_1_1101_9908_9230,M02171_86_000000000-D0H8A_1_1101_10532_9073,M02171_86_000000000-D0H8A_1_1101_16542_13147,M02171_86_000000000-D0H8A_1_1101_2967_13181,M02171_86_000000000-D0H8A_1_1101_20658_12727,M02171_86_000000000-D0H8A_1_1101_11362_12788,M02171_86_000000000-D0H8A_1_1101_28431_13856,M02171_86_000000000-D0H8A_1_1101_17421_11498,M02171_86_000000000-D0H8A_1_1101_18452_11715,M02171_86_000000000-D0H8A_1_1101_17439_11608,M02171_86_000000000-D0H8A_1_1101_16928_11337,M02171_86_000000000-D0H8A_1_1101_21329_12428,M02171_86_000000000-D0H8A_1_1101_13930_12543,M02171_86_000000000-D0H8A_1_1101_27566_11847,M02171_86_000000000-D0H8A_1_1101_14395_4079,M02171_86_000000000-D0H8A_1_1101_13057_3936,M02171_86_000000000-D0H8A_1_1101_25000_4999,M02171_86_000000000-D0H8A_1_1101_24902_4974,M02171_86_000000000-D0H8A_1_1101_11560_4640,M02171_86_000000000-D0H8A_1_1101_24296_4853,M02171_86_000000000-D0H8A_1_1101_20582_2261,M02171_86_000000000-D0H8A_1_1101_12798_2158,M02171_86_000000000-D0H8A_1_1101_20915_3018,M02171_86_000000000-D0H8A_1_1101_12704_2851,M02171_86_000000000-D0H8A_1_1101_14666_7265,M02171_86_000000000-D0H8A_1_1101_7276_6875,M02171_86_000000000-D0H8A_1_1101_13897_6943,M02171_86_000000000-D0H8A_1_1101_21042_7045,M02171_86_000000000-D0H8A_1_1101_24748_7876,M02171_86_000000000-D0H8A_1_1101_14235_7695,M02171_86_000000000-D0H8A_1_1101_25184_7507,M02171_86_000000000-D0H8A_1_1101_6199_6017,M02171_86_000000000-D0H8A_1_1101_11418_5954,M02171_86_000000000-D0H8A_1_1101_19197_6133,M02171_86_000000000-D0H8A_1_1101_13698_6420,M02171_86_000000000-D0H8A_1_1101_12872_6355,M02171_86_000000000-D0H8A_1_1101_17150_6328,M02171_86_000000000-D0H8A_1_1101_8060_21871,M02171_86_000000000-D0H8A_1_1101_18810_22169,M02171_86_000000000-D0H8A_1_1101_19813_21723,M02171_86_000000000-D0H8A_1_1101_6121_21662,M02171_86_000000000-D0H8A_1_1101_25607_20313,M02171_86_000000000-D0H8A_1_1101_8253_20116,M02171_86_000000000-D0H8A_1_1101_18153_20022,M02171_86_000000000-D0H8A_1_1101_19656_21342,M02171_86_000000000-D0H8A_1_1101_16925_21049,M02171_86_000000000-D0H8A_1_1101_21681_25889,M02171_86_000000000-D0H8A_1_1101_15414_25261,M02171_86_000000000-D0H8A_1_1101_13521_25436,M02171_86_000000000-D0H8A_1_1101_18564_26968,M02171_86_000000000-D0H8A_1_1101_22906_26220,M02171_86_000000000-D0H8A_1_1101_17480_26111,M02171_86_000000000-D0H8A_1_1101_5258_23403,M02171_86_000000000-D0H8A_1_1101_2432_15600,M02171_86_000000000-D0H8A_1_1101_2440_15584,M02171_86_000000000-D0H8A_1_1101_21188_15692,M02171_86_000000000-D0H8A_1_1101_25981_16311,M02171_86_000000000-D0H8A_1_1101_21959_14491,M02171_86_000000000-D0H8A_1_1101_2793_14173,M02171_86_000000000-D0H8A_1_1101_24716_14378,M02171_86_000000000-D0H8A_1_1101_9901_18990,M02171_86_000000000-D0H8A_1_1101_21872_19022,M02171_86_000000000-D0H8A_1_1101_13820_18511,M02171_86_000000000-D0H8A_1_1101_3109_18456,M02171_86_000000000-D0H8A_1_1101_10498_19920,M02171_86_000000000-D0H8A_1_1101_27923_19897,M02171_86_000000000-D0H8A_1_1101_28912_19477,M02171_86_000000000-D0H8A_1_1101_26731_17219,M02171_86_000000000-D0H8A_1_1101_11983_18175,M02171_86_000000000-D0H8A_1_1101_18234_18047 is not in your count table. Please correct.
[ERROR]: 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is not in your count table. Please correct.

mothur > quit()
2

Could you retry running that command in 1.39.5 with 1 or 2 processors? Alternatively, could you go back to the last screen.seqs command and rerun that and the subsequent commands?

Pat

3

If Pat’s suggestions don’t resolve the issue, can you send me your log file, as well as Miseq2nd_R1.trim.contigs.fasta and Miseq2nd_R1.contigs.groups, so I can track down the issue for you?

4

Thanks so much for the help, Dr. Pat and Dr. Westcott. Really appreciate the Mothur Team’s help along the study. Got fixed! Everything is running well now. May I know what is the reason for that? Because I ran this on university’s supercomputer system. So I always set it as processors=16.

And I have another question, is there a space limit for the data running? Because I face a situation, when I ran a raw 16S Miseq data(forward.fastq5G, and reverse fastq 5G) on the university’s super computer(I have 1T storage space, which I thought is enough for data running) in two ways, one is directly under my folder of university’s computer, another one is submit it as a job file run. Everything is almost the same before doing align.seqs().

However, the alignment result directly under my folder is 14G, while job file run result is 21G. In order to identify this, I also run it on my own desktop(windows version), I also got 14G alignment result. So far, I can not believe which result shall I believe.