Chimera.vsearch not removing chimeras - screen.seqs to blame?

Dear Mothur team

In the early workflow, the Mothur SOP has users perform make.contigs followed by summary.seqs. The next instruction is then to filter the sequences based on specified criteria (max length, and so on) using either make.contigs again or screen.seqs. Mothur advises to use screen.seqs over running make.contigs a second time. However, I found that when running screen.seqs at this stage, later on in the SOP, the chimera.vserach command does not remove chimeras from the dataset (despite seemingly detecting them).

I troubleshooted this back to the make.contigs/screen.seqs commands and found that when running make.contigs a second time (with maxlength, maxhomop, maxambig filters, rather than screen.seqs) it resolves the problem.

As aforementioned, when using screen.seqs, the chimera.vserach command detects identical number of chimeras to when running make.contigs (the second time), e.g. iH2O4 and iMC04 which have 28 and 318 chimeras, respectively, in both log files.

Furthermore, when using the screen.seqs command, following chimera.vsearch I get the error “Error in reading your fastafile, at position -1. Blank name.” Perhaps this is what’s preventing chimera removal?

Of course, it may be user error and I may have incorrect syntax although I believe I ran identical scripts both times (apart from the extra “good” missing from the file names in one of the workflows due to running make.contigs rather than screen.seqs).

Happy to send my log files/code to your inbox if it helps.

Best regards
James

Hi James,

Could you post the output of running summary.seqs after running screen.seqs? I suspect the problem you’re having is because you aren’t using the count file in all of the steps (perhaps in unique.seqs?

Pat

Dear Pat

Thanks for your response. Please find the code below.

mothur > 
screen.seqs(fasta=stability.trim.contigs.fasta, count=stability.contigs.count_table, maxambig=0, maxlength=275, maxhomop=8)

Using 16 processors.

It took 38 secs to screen 9723305 sequences, removed 4179109.

/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.bad.accnos.temp, count=stability.contigs.count_table)
Removed 4179109 sequences from stability.contigs.count_table.

Output File Names:
stability.contigs.pick.count_table

/******************************************/

Output File Names:
stability.trim.contigs.good.fasta
stability.trim.contigs.bad.accnos
stability.contigs.good.count_table


It took 244 secs to screen 9723305 sequences.

mothur > 
summary.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.good.count_table)

Using 16 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	249	249	0	3	1
2.5%-tile:	1	252	252	0	3	138605
25%-tile:	1	253	253	0	3	1386050
Median: 	1	253	253	0	4	2772099
75%-tile:	1	253	253	0	4	4158148
97.5%-tile:	1	254	254	0	6	5405592
Maximum:	1	275	275	0	8	5544196
Mean:	1	252	252	0	3
 of unique seqs:	5544196
total # of seqs:	5544196

It took 120 secs to summarize 5544196 sequences.

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


mothur > 
unique.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.good.count_table)
5544196	298046

Output File Names: 
stability.trim.contigs.good.unique.fasta
stability.trim.contigs.good.count_table


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

Hi James,

I don’t see the output of running that final summary.seqs command. Can you post that?

summary.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.good.count_table)

If you could post the rest of the pipeline through what you’re seeing with chimera checkin that would be great too.

Pat

Hi Pat,

In the above I’ve provided screen.seqs, summary.seqs and unique.seqs, all sequential in the Mothur pipeline.

Below I will provide the output of summary.seqs following the unique.seqs command. I will also provide output of chimera.vsearch and the sequential summary.seqs.

unique.seqs / summary.seqs:

mothur > 
unique.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.good.count_table)
5544196	298046

Output File Names: 
stability.trim.contigs.good.unique.fasta
stability.trim.contigs.good.count_table


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

Using 16 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	249	249	0	3	1
2.5%-tile:	1	252	252	0	3	138605
25%-tile:	1	253	253	0	3	1386050
Median: 	1	253	253	0	4	2772099
75%-tile:	1	253	253	0	4	4158148
97.5%-tile:	1	254	254	0	6	5405592
Maximum:	1	275	275	0	8	5544196
Mean:	1	252	252	0	3
# of unique seqs:	298046
total # of seqs:	5544196

It took 7 secs to summarize 5544196 sequences.

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

chimera.vsearch / summary.seqs

mothur > 
chimera.vsearch(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t)

Using 16 processors.
Checking sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta ...

/******************************************/
Splitting by sample: 

Using 16 processors.

Selecting sequences for groups D39D_25th_3_2572_CTL_XX-D39E_25th_3_2572_CTL_XX-D39F_25th_3_2572_CTL_XX-D40D_25th_4_2769_CTL_XX-D40E_25th_4_2769_CTL_XX-D40F_25th_4_2769_CTL_XX-D41D_25th_5_2624_XOS_Hi-D41E_25th_5_2624_XOS_Hi-D41F_25th_5_2624_XOS_Hi-D42D_25th_6_2549_XOS_Hi


Selecting sequences for groups D15F_24th_15_2472_XOS_Lo-D16D_24th_16_2489_XOS_Lo-D16E_24th_16_2489_XOS_Lo-D16F_24th_16_2489_XOS_Lo-D17D_24th_17_2529_XOS_Hi-D17E_24th_17_2529_XOS_Hi-D17F_24th_17_2529_XOS_Hi-D18D_24th_18_2750_XOS_Hi-D18E_24th_18_2750_XOS_Hi-D18F_24th_18_2750_XOS_Hi


Selecting sequences for groups D20E_24th_20_2773_XOS_Lo-D20F_24th_20_2773_XOS_Lo-D21D_24th_21_2786_XOS_Lo-D21E_24th_21_2786_XOS_Lo-D21F_24th_21_2786_XOS_Lo-D22D_24th_22_2632_XOS_Hi-D22E_24th_22_2632_XOS_Hi-D22F_24th_22_2632_XOS_Hi-D23D_24th_23_2692_CTL_XX-D23E_24th_23_2692_CTL_XX


Selecting sequences for groups D44D_25th_8_2676_XOS_Lo-D44E_25th_8_2676_XOS_Lo-D44F_25th_8_2676_XOS_Lo-D45D_25th_9_2782_XOS_Lo-D45E_25th_9_2782_XOS_Lo-D45F_25th_9_2782_XOS_Lo-D46D_25th_10_2485_XOS_Hi-D46E_25th_10_2485_XOS_Hi-D46F_25th_10_2485_XOS_Hi-D47D_25th_11_2673_XOS_Lo


Selecting sequences for groups D64F_25th_28_2828_CTL_XX-D65D_25th_29_2586_XOS_Hi-D65E_25th_29_2586_XOS_Hi-D65F_25th_29_2586_XOS_Hi-D66D_25th_30_2569_XOS_Hi-D66E_25th_30_2569_XOS_Hi-D66F_25th_30_2569_XOS_Hi-D67D_25th_31_2717_CTL_XX-D67E_25th_31_2717_CTL_XX-D67F_25th_31_2717_CTL_XX


Selecting sequences for groups D25D_24th_25_2652_XOS_Hi-D25E_24th_25_2652_XOS_Hi-D25F_24th_25_2652_XOS_Hi-D26D_24th_26_2715_CTL_XX-D26E_24th_26_2715_CTL_XX-D26F_24th_26_2715_CTL_XX-D27D_24th_27_2770_XOS_Lo-D27E_24th_27_2770_XOS_Lo-D27F_24th_27_2770_XOS_Lo-D28D_24th_28_2723_CTL_XX


Selecting sequences for groups D69F_25th_33_2753_XOS_Lo-D70D_25th_34_2686_XOS_Lo-D71D_25th_35_2486_CTL_XX-D71E_25th_35_2486_CTL_XX-D71F_25th_35_2486_CTL_XX-D72D_25th_36_2727_XOS_Lo-D72E_25th_36_2727_XOS_Lo-D72F_25th_36_2727_XOS_Lo-H2O1-H2O2


Selecting sequences for groups D06E_24th_06_2674_XOS_Hi-D06F_24th_06_2674_XOS_Hi-D07D_24th_07_2857_CTL_XX-D07E_24th_07_2857_CTL_XX-D07F_24th_07_2857_CTL_XX-D08D_24th_08_2677_XOS_Lo-D08E_24th_08_2677_XOS_Lo-D08F_24th_08_2677_XOS_Lo-D09D_24th_09_2614_XOS_Lo-D09E_24th_09_2614_XOS_Lo


Selecting sequences for groups D01D_24th_01_2805_XOS_Hi-D02D_24th_02_2570_XOS_Lo-D02E_24th_02_2570_XOS_Lo-D02F_24th_02_2570_XOS_Lo-D03D_24th_03_2601_CTL_XX-D03E_24th_03_2601_CTL_XX-D03F_24th_03_2601_CTL_XX-D04D_24th_04_2768_CTL_XX-D04E_24th_04_2768_CTL_XX-D04F_24th_04_2768_CTL_XX


Selecting sequences for groups D11D_24th_11_2879_XOS_Lo-D11E_24th_11_2879_XOS_Lo-D11F_24th_11_2879_XOS_Lo-D12D_24th_12_2778_CTL_XX-D12E_24th_12_2778_CTL_XX-D12F_24th_12_2778_CTL_XX-D13D_24th_13_2713_XOS_Hi-D13E_24th_13_2713_XOS_Hi-D13F_24th_13_2713_XOS_Hi-D14D_24th_14_2665_CTL_XX


Selecting sequences for groups D54F_25th_18_2656_XOS_Hi-D55D_25th_19_2850_CTL_XX-D55E_25th_19_2850_CTL_XX-D55F_25th_19_2850_CTL_XX-D56D_25th_20_2833_XOS_Lo-D56E_25th_20_2833_XOS_Lo-D56F_25th_20_2833_XOS_Lo-D57D_25th_21_2733_XOS_Lo-D57E_25th_21_2733_XOS_Lo-D57F_25th_21_2733_XOS_Lo


Selecting sequences for groups D34E_24th_34_2700_XOS_Lo-D34F_24th_34_2700_XOS_Lo-D35D_24th_35_2507_CTL_XX-D35E_24th_35_2507_CTL_XX-D35F_24th_35_2507_CTL_XX-D36D_24th_36_2840_XOS_Lo-D36E_24th_36_2840_XOS_Lo-D36F_24th_36_2840_XOS_Lo-D37D_25th_1_2874_XOS_Hi-D37E_25th_1_2874_XOS_Hi


Selecting sequences for groups D59F_25th_23_2867_CTL_XX-D60D_25th_24_2556_CTL_XX-D60E_25th_24_2556_CTL_XX-D60F_25th_24_2556_CTL_XX-D61D_25th_25_2870_XOS_Hi-D61E_25th_25_2870_XOS_Hi-D61F_25th_25_2870_XOS_Hi-D62D_25th_26_2458_CTL_XX-D62E_25th_26_2458_CTL_XX-D62F_25th_26_2458_CTL_XX


Selecting sequences for groups D49D_25th_13_2605_XOS_Hi-D50D_25th_14_2837_CTL_XX-D50E_25th_14_2837_CTL_XX-D50F_25th_14_2837_CTL_XX-D51D_25th_15_2591_XOS_Lo-D51E_25th_15_2591_XOS_Lo-D51F_25th_15_2591_XOS_Lo-D52D_25th_16_2856_XOS_Lo-D52E_25th_16_2856_XOS_Lo-D52F_25th_16_2856_XOS_Lo


Selecting sequences for groups D29F_24th_29_2567_XOS_Hi-D30D_24th_30_2705_XOS_Hi-D30E_24th_30_2705_XOS_Hi-D30F_24th_30_2705_XOS_Hi-D31D_24th_31_2861_CTL_XX-D31E_24th_31_2861_CTL_XX-D31F_24th_31_2861_CTL_XX-D32D_24th_32_2696_XOS_Hi-D32E_24th_32_2696_XOS_Hi-D32F_24th_32_2696_XOS_Hi


Selecting sequences for groups MC03-MC05-Undetermined-VH2O-i01E_24th_01_2805_XOS_Hi-i01F_24th_01_2805_XOS_Hi-i16D_24th_16_2489_XOS_Lo-i43F_25th_7_2635_CTL_XX-i49E_25th_13_2605_XOS_Hi-i49F_25th_13_2605_XOS_Hi

Selected 958 sequences from D39D_25th_3_2572_CTL_XX.
Selected 1260 sequences from D39E_25th_3_2572_CTL_XX.
Selected 1748 sequences from D39F_25th_3_2572_CTL_XX.
Selected 769 sequences from D40D_25th_4_2769_CTL_XX.
Selected 1114 sequences from D40E_25th_4_2769_CTL_XX.
Selected 1091 sequences from D40F_25th_4_2769_CTL_XX.
Selected 440 sequences from D41D_25th_5_2624_XOS_Hi.
Selected 444 sequences from D41E_25th_5_2624_XOS_Hi.
Selected 500 sequences from D41F_25th_5_2624_XOS_Hi.
Selected 719 sequences from D42D_25th_6_2549_XOS_Hi.

Selecting sequences for groups D42E_25th_6_2549_XOS_Hi-D42F_25th_6_2549_XOS_Hi-D43D_25th_7_2635_CTL_XX-D43E_25th_7_2635_CTL_XX-D43F_25th_7_2635_CTL_XX

Selected 612 sequences from D25D_24th_25_2652_XOS_Hi.
Selected 837 sequences from D25E_24th_25_2652_XOS_Hi.
Selected 975 sequences from D25F_24th_25_2652_XOS_Hi.
Selected 990 sequences from D26D_24th_26_2715_CTL_XX.
Selected 1136 sequences from D26E_24th_26_2715_CTL_XX.
Selected 283 sequences from D26F_24th_26_2715_CTL_XX.
Selected 1123 sequences from D27D_24th_27_2770_XOS_Lo.
Selected 2184 sequences from D27E_24th_27_2770_XOS_Lo.
Selected 2140 sequences from D27F_24th_27_2770_XOS_Lo.
Selected 380 sequences from D28D_24th_28_2723_CTL_XX.
Selected 1003 sequences from D44D_25th_8_2676_XOS_Lo.
Selected 1379 sequences from D44E_25th_8_2676_XOS_Lo.
Selected 2151 sequences from D44F_25th_8_2676_XOS_Lo.
Selected 533 sequences from D45D_25th_9_2782_XOS_Lo.
Selected 940 sequences from D45E_25th_9_2782_XOS_Lo.
Selected 1066 sequences from D45F_25th_9_2782_XOS_Lo.
Selected 305 sequences from D46D_25th_10_2485_XOS_Hi.
Selected 1106 sequences from D46E_25th_10_2485_XOS_Hi.
Selected 832 sequences from D46F_25th_10_2485_XOS_Hi.
Selected 830 sequences from D47D_25th_11_2673_XOS_Lo.

Selecting sequences for groups D28E_24th_28_2723_CTL_XX-D28F_24th_28_2723_CTL_XX-D29D_24th_29_2567_XOS_Hi-D29E_24th_29_2567_XOS_Hi

Selected 1338 sequences from D59F_25th_23_2867_CTL_XX.
Selected 1043 sequences from D60D_25th_24_2556_CTL_XX.
Selected 1126 sequences from D60E_25th_24_2556_CTL_XX.
Selected 928 sequences from D60F_25th_24_2556_CTL_XX.
Selected 1120 sequences from D61D_25th_25_2870_XOS_Hi.
Selected 1017 sequences from D61E_25th_25_2870_XOS_Hi.
Selected 1166 sequences from D61F_25th_25_2870_XOS_Hi.
Selected 1189 sequences from D62D_25th_26_2458_CTL_XX.
Selected 1728 sequences from D62E_25th_26_2458_CTL_XX.
Selected 1953 sequences from D62F_25th_26_2458_CTL_XX.

Selecting sequences for groups D47E_25th_11_2673_XOS_Lo-D47F_25th_11_2673_XOS_Lo-D48D_25th_12_2730_CTL_XX-D48E_25th_12_2730_CTL_XX-D48F_25th_12_2730_CTL_XX

Selected 1030 sequences from D06E_24th_06_2674_XOS_Hi.
Selected 700 sequences from D06F_24th_06_2674_XOS_Hi.
Selected 1129 sequences from D07D_24th_07_2857_CTL_XX.
Selected 1131 sequences from D07E_24th_07_2857_CTL_XX.
Selected 1093 sequences from D07F_24th_07_2857_CTL_XX.
Selected 962 sequences from D08D_24th_08_2677_XOS_Lo.
Selected 1121 sequences from D08E_24th_08_2677_XOS_Lo.
Selected 846 sequences from D08F_24th_08_2677_XOS_Lo.
Selected 775 sequences from D09D_24th_09_2614_XOS_Lo.
Selected 579 sequences from D09E_24th_09_2614_XOS_Lo.
Selected 1099 sequences from D64F_25th_28_2828_CTL_XX.
Selected 913 sequences from D65D_25th_29_2586_XOS_Hi.
Selected 1005 sequences from D65E_25th_29_2586_XOS_Hi.
Selected 845 sequences from D65F_25th_29_2586_XOS_Hi.
Selected 661 sequences from D66D_25th_30_2569_XOS_Hi.
Selected 1071 sequences from D66E_25th_30_2569_XOS_Hi.
Selected 1422 sequences from D66F_25th_30_2569_XOS_Hi.
Selected 1053 sequences from D67D_25th_31_2717_CTL_XX.
Selected 890 sequences from D20E_24th_20_2773_XOS_Lo.
Selected 1202 sequences from D67E_25th_31_2717_CTL_XX.
Selected 1888 sequences from D67F_25th_31_2717_CTL_XX.
Selected 954 sequences from D20F_24th_20_2773_XOS_Lo.
Selected 1249 sequences from D21D_24th_21_2786_XOS_Lo.
Selected 1321 sequences from D21E_24th_21_2786_XOS_Lo.
Selected 1131 sequences from D21F_24th_21_2786_XOS_Lo.
Selected 922 sequences from D22D_24th_22_2632_XOS_Hi.
Selected 1548 sequences from D22E_24th_22_2632_XOS_Hi.
Selected 1135 sequences from D22F_24th_22_2632_XOS_Hi.
Selected 573 sequences from D23D_24th_23_2692_CTL_XX.
Selected 722 sequences from D23E_24th_23_2692_CTL_XX.
Selected 930 sequences from D15F_24th_15_2472_XOS_Lo.
Selected 20 sequences from D16D_24th_16_2489_XOS_Lo.
Selected 1250 sequences from D16E_24th_16_2489_XOS_Lo.
Selected 1006 sequences from D16F_24th_16_2489_XOS_Lo.
Selected 814 sequences from D17D_24th_17_2529_XOS_Hi.
Selected 1302 sequences from D17E_24th_17_2529_XOS_Hi.
Selected 1264 sequences from D17F_24th_17_2529_XOS_Hi.
Selected 643 sequences from D18D_24th_18_2750_XOS_Hi.
Selected 882 sequences from D18E_24th_18_2750_XOS_Hi.
Selected 572 sequences from D18F_24th_18_2750_XOS_Hi.

Selecting sequences for groups D63D_25th_27_2513_XOS_Lo-D63E_25th_27_2513_XOS_Lo-D63F_25th_27_2513_XOS_Lo-D64D_25th_28_2828_CTL_XX-D64E_25th_28_2828_CTL_XX


Selecting sequences for groups D23F_24th_23_2692_CTL_XX-D24D_24th_24_2530_CTL_XX-D24E_24th_24_2530_CTL_XX-D24F_24th_24_2530_CTL_XX


Selecting sequences for groups D68D_25th_32_2621_XOS_Hi-D68E_25th_32_2621_XOS_Hi-D68F_25th_32_2621_XOS_Hi-D69D_25th_33_2753_XOS_Lo-D69E_25th_33_2753_XOS_Lo


Selecting sequences for groups D09F_24th_09_2614_XOS_Lo-D10D_24th_10_2553_XOS_Hi-D10E_24th_10_2553_XOS_Hi-D10F_24th_10_2553_XOS_Hi


Selecting sequences for groups D19D_24th_19_2582_CTL_XX-D19E_24th_19_2582_CTL_XX-D19F_24th_19_2582_CTL_XX-D20D_24th_20_2773_XOS_Lo

Selected 741 sequences from D01D_24th_01_2805_XOS_Hi.
Selected 1156 sequences from D02D_24th_02_2570_XOS_Lo.
Selected 1496 sequences from D02E_24th_02_2570_XOS_Lo.
Selected 1297 sequences from D02F_24th_02_2570_XOS_Lo.
Selected 607 sequences from D03D_24th_03_2601_CTL_XX.
Selected 908 sequences from D03E_24th_03_2601_CTL_XX.
Selected 799 sequences from D03F_24th_03_2601_CTL_XX.
Selected 459 sequences from D04D_24th_04_2768_CTL_XX.
Selected 1409 sequences from D04E_24th_04_2768_CTL_XX.
Selected 1386 sequences from D04F_24th_04_2768_CTL_XX.
Selected 1014 sequences from D69F_25th_33_2753_XOS_Lo.
Selected 1611 sequences from D70D_25th_34_2686_XOS_Lo.
Selected 523 sequences from D71D_25th_35_2486_CTL_XX.
Selected 1166 sequences from D71E_25th_35_2486_CTL_XX.
Selected 1169 sequences from D71F_25th_35_2486_CTL_XX.
Selected 1050 sequences from D72D_25th_36_2727_XOS_Lo.
Selected 996 sequences from D72E_25th_36_2727_XOS_Lo.
Selected 913 sequences from D72F_25th_36_2727_XOS_Lo.
Selected 23 sequences from H2O1.
Selected 24 sequences from H2O2.
Selected 673 sequences from D54F_25th_18_2656_XOS_Hi.
Selected 716 sequences from D55D_25th_19_2850_CTL_XX.
Selected 1513 sequences from D55E_25th_19_2850_CTL_XX.
Selected 1744 sequences from D55F_25th_19_2850_CTL_XX.
Selected 742 sequences from D56D_25th_20_2833_XOS_Lo.
Selected 1054 sequences from D56E_25th_20_2833_XOS_Lo.
Selected 866 sequences from D56F_25th_20_2833_XOS_Lo.
Selected 533 sequences from D57D_25th_21_2733_XOS_Lo.
Selected 954 sequences from D57E_25th_21_2733_XOS_Lo.
Selected 1532 sequences from D57F_25th_21_2733_XOS_Lo.
Selected 436 sequences from D11D_24th_11_2879_XOS_Lo.
Selected 680 sequences from D11E_24th_11_2879_XOS_Lo.
Selected 765 sequences from D11F_24th_11_2879_XOS_Lo.
Selected 1254 sequences from D12D_24th_12_2778_CTL_XX.
Selected 1329 sequences from D12E_24th_12_2778_CTL_XX.
Selected 1541 sequences from D12F_24th_12_2778_CTL_XX.
Selected 1076 sequences from D13D_24th_13_2713_XOS_Hi.
Selected 1163 sequences from D13E_24th_13_2713_XOS_Hi.
Selected 1422 sequences from D13F_24th_13_2713_XOS_Hi.
Selected 783 sequences from D14D_24th_14_2665_CTL_XX.

Selecting sequences for groups H2O3-H2O5-LTH2O-MC01-MC02


Selecting sequences for groups D05D_24th_05_2835_XOS_Hi-D05E_24th_05_2835_XOS_Hi-D05F_24th_05_2835_XOS_Hi-D06D_24th_06_2674_XOS_Hi


Selecting sequences for groups D14E_24th_14_2665_CTL_XX-D14F_24th_14_2665_CTL_XX-D15D_24th_15_2472_XOS_Lo-D15E_24th_15_2472_XOS_Lo

Selected 975 sequences from D34E_24th_34_2700_XOS_Lo.
Selected 1054 sequences from D34F_24th_34_2700_XOS_Lo.
Selected 935 sequences from D35D_24th_35_2507_CTL_XX.
Selected 818 sequences from D35E_24th_35_2507_CTL_XX.
Selected 951 sequences from D35F_24th_35_2507_CTL_XX.
Selected 1198 sequences from D36D_24th_36_2840_XOS_Lo.
Selected 1340 sequences from D36E_24th_36_2840_XOS_Lo.
Selected 1482 sequences from D36F_24th_36_2840_XOS_Lo.
Selected 685 sequences from D37D_25th_1_2874_XOS_Hi.
Selected 1477 sequences from D37E_25th_1_2874_XOS_Hi.
Selected 1134 sequences from D49D_25th_13_2605_XOS_Hi.
Selected 69 sequences from D50D_25th_14_2837_CTL_XX.
Selected 1858 sequences from D50E_25th_14_2837_CTL_XX.
Selected 1603 sequences from D50F_25th_14_2837_CTL_XX.
Selected 2321 sequences from D51D_25th_15_2591_XOS_Lo.
Selected 1722 sequences from D51E_25th_15_2591_XOS_Lo.
Selected 1929 sequences from D51F_25th_15_2591_XOS_Lo.
Selected 906 sequences from D52D_25th_16_2856_XOS_Lo.
Selected 1131 sequences from D52E_25th_16_2856_XOS_Lo.
Selected 996 sequences from D52F_25th_16_2856_XOS_Lo.

Selecting sequences for groups D58D_25th_22_2628_XOS_Hi-D58E_25th_22_2628_XOS_Hi-D58F_25th_22_2628_XOS_Hi-D59D_25th_23_2867_CTL_XX-D59E_25th_23_2867_CTL_XX

Selected 1354 sequences from D29F_24th_29_2567_XOS_Hi.
Selected 2333 sequences from D30D_24th_30_2705_XOS_Hi.
Selected 2430 sequences from D30E_24th_30_2705_XOS_Hi.
Selected 2335 sequences from D30F_24th_30_2705_XOS_Hi.
Selected 359 sequences from D31D_24th_31_2861_CTL_XX.
Selected 534 sequences from D31E_24th_31_2861_CTL_XX.
Selected 144 sequences from D31F_24th_31_2861_CTL_XX.
Selected 565 sequences from D32D_24th_32_2696_XOS_Hi.
Selected 1003 sequences from D32E_24th_32_2696_XOS_Hi.
Selected 1225 sequences from D32F_24th_32_2696_XOS_Hi.

Selecting sequences for groups D53D_25th_17_2675_XOS_Hi-D53E_25th_17_2675_XOS_Hi-D53F_25th_17_2675_XOS_Hi-D54D_25th_18_2656_XOS_Hi-D54E_25th_18_2656_XOS_Hi


Selecting sequences for groups D37F_25th_1_2874_XOS_Hi-D38D_25th_2_2812_XOS_Lo-D38E_25th_2_2812_XOS_Lo-D38F_25th_2_2812_XOS_Lo


Selecting sequences for groups D33D_24th_33_2480_XOS_Lo-D33E_24th_33_2480_XOS_Lo-D33F_24th_33_2480_XOS_Lo-D34D_24th_34_2700_XOS_Lo

Selected 321 sequences from MC03.
Selected 74 sequences from MC05.
Selected 9804 sequences from Undetermined.
Selected 29 sequences from VH2O.
Selected 976 sequences from i01E_24th_01_2805_XOS_Hi.
Selected 1207 sequences from i01F_24th_01_2805_XOS_Hi.
Selected 846 sequences from i16D_24th_16_2489_XOS_Lo.
Selected 1193 sequences from i43F_25th_7_2635_CTL_XX.
Selected 1859 sequences from i49E_25th_13_2605_XOS_Hi.
Selected 1567 sequences from i49F_25th_13_2605_XOS_Hi.

Selecting sequences for groups i50D_25th_14_2837_CTL_XX-i70E_25th_34_2686_XOS_Lo-i70F_25th_34_2686_XOS_Lo-iH2O4-iMC04

Selected 568 sequences from D28E_24th_28_2723_CTL_XX.
Selected 270 sequences from D28F_24th_28_2723_CTL_XX.
Selected 1424 sequences from D29D_24th_29_2567_XOS_Hi.
Selected 1339 sequences from D29E_24th_29_2567_XOS_Hi.
Selected 758 sequences from D42E_25th_6_2549_XOS_Hi.
Selected 701 sequences from D42F_25th_6_2549_XOS_Hi.
Selected 722 sequences from D43D_25th_7_2635_CTL_XX.
Selected 842 sequences from D43E_25th_7_2635_CTL_XX.
Selected 32 sequences from D43F_25th_7_2635_CTL_XX.
Selected 1119 sequences from D47E_25th_11_2673_XOS_Lo.
Selected 948 sequences from D47F_25th_11_2673_XOS_Lo.
Selected 1003 sequences from D48D_25th_12_2730_CTL_XX.
Selected 870 sequences from D48E_25th_12_2730_CTL_XX.
Selected 728 sequences from D48F_25th_12_2730_CTL_XX.
Selected 486 sequences from D63D_25th_27_2513_XOS_Lo.
Selected 1080 sequences from D63E_25th_27_2513_XOS_Lo.
Selected 948 sequences from D63F_25th_27_2513_XOS_Lo.
Selected 845 sequences from D64D_25th_28_2828_CTL_XX.
Selected 972 sequences from D64E_25th_28_2828_CTL_XX.
Selected 282 sequences from D68D_25th_32_2621_XOS_Hi.
Selected 896 sequences from D68E_25th_32_2621_XOS_Hi.
Selected 783 sequences from D68F_25th_32_2621_XOS_Hi.
Selected 567 sequences from D69D_25th_33_2753_XOS_Lo.
Selected 836 sequences from D69E_25th_33_2753_XOS_Lo.
Selected 716 sequences from D23F_24th_23_2692_CTL_XX.
Selected 609 sequences from D24D_24th_24_2530_CTL_XX.
Selected 1206 sequences from D24E_24th_24_2530_CTL_XX.
Selected 1142 sequences from D24F_24th_24_2530_CTL_XX.
Selected 428 sequences from D09F_24th_09_2614_XOS_Lo.
Selected 1787 sequences from D10D_24th_10_2553_XOS_Hi.
Selected 1826 sequences from D10E_24th_10_2553_XOS_Hi.
Selected 1937 sequences from D10F_24th_10_2553_XOS_Hi.
Selected 1276 sequences from D19D_24th_19_2582_CTL_XX.
Selected 1175 sequences from D19E_24th_19_2582_CTL_XX.
Selected 1420 sequences from D19F_24th_19_2582_CTL_XX.
Selected 618 sequences from D20D_24th_20_2773_XOS_Lo.
Selected 19 sequences from H2O3.
Selected 13 sequences from H2O5.
Selected 33 sequences from LTH2O.
Selected 446 sequences from MC01.
Selected 483 sequences from MC02.
Selected 528 sequences from D14E_24th_14_2665_CTL_XX.
Selected 554 sequences from D14F_24th_14_2665_CTL_XX.
Selected 831 sequences from D15D_24th_15_2472_XOS_Lo.
Selected 912 sequences from D15E_24th_15_2472_XOS_Lo.
Selected 330 sequences from D53D_25th_17_2675_XOS_Hi.
Selected 214 sequences from D53E_25th_17_2675_XOS_Hi.
Selected 443 sequences from D53F_25th_17_2675_XOS_Hi.
Selected 302 sequences from D54D_25th_18_2656_XOS_Hi.
Selected 551 sequences from D54E_25th_18_2656_XOS_Hi.
Selected 1363 sequences from D05D_24th_05_2835_XOS_Hi.
Selected 1430 sequences from D05E_24th_05_2835_XOS_Hi.
Selected 1574 sequences from D05F_24th_05_2835_XOS_Hi.
Selected 843 sequences from D06D_24th_06_2674_XOS_Hi.
Selected 1206 sequences from i50D_25th_14_2837_CTL_XX.
Selected 1291 sequences from i70E_25th_34_2686_XOS_Lo.
Selected 1832 sequences from i70F_25th_34_2686_XOS_Lo.
Selected 28 sequences from iH2O4.
Selected 318 sequences from iMC04.
Selected 881 sequences from D37F_25th_1_2874_XOS_Hi.
Selected 1176 sequences from D38D_25th_2_2812_XOS_Lo.
Selected 1067 sequences from D38E_25th_2_2812_XOS_Lo.
Selected 1202 sequences from D38F_25th_2_2812_XOS_Lo.
Selected 695 sequences from D58D_25th_22_2628_XOS_Hi.
Selected 1792 sequences from D58E_25th_22_2628_XOS_Hi.
Selected 1362 sequences from D58F_25th_22_2628_XOS_Hi.
Selected 1854 sequences from D59D_25th_23_2867_CTL_XX.
Selected 1380 sequences from D59E_25th_23_2867_CTL_XX.
Selected 412 sequences from D33D_24th_33_2480_XOS_Lo.
Selected 764 sequences from D33E_24th_33_2480_XOS_Lo.
Selected 957 sequences from D33F_24th_33_2480_XOS_Lo.
Selected 1152 sequences from D34D_24th_34_2700_XOS_Lo.

It took 11 seconds to split the dataset by sample.
/******************************************/
Error in reading your fastafile, at position -1. Blank name.
It took 3 secs to check 0 sequences.


mothur > 
summary.seqs(fasta=current, count=current)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.

Using 16 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	490	231	0	3	1
2.5%-tile:	1	490	252	0	3	136825
25%-tile:	1	490	253	0	3	1368245
Median: 	1	490	253	0	4	2736490
75%-tile:	1	490	253	0	4	4104734
97.5%-tile:	1	490	253	0	6	5336154
Maximum:	1	490	265	0	8	5472978
Mean:	1	490	252	0	3
# of unique seqs:	93742
total # of seqs:	5472978

It took 2 secs to summarize 5472978 sequences.

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.summary

James

Hi James,
I am happy to help. What version of mothur are you running? If it’s not 1.48.1, are you seeing this issue with our most current release, 1.48.1 release? If you are, could you send your input files and log files to mothur.westcott@gmail.com so I can troubleshoot the issue for you?
Thanks,
Sarah Westcott

Hi Sarah,
I am running 1.48.1 and have sent logfiles to the email address.
James

Hi James,
Thanks for sending your files. I ran them on our Windows test machine and was unable to reproduce the issue you are having. We have had similar bug reports occur when a user runs out of disk space to write the output files, which may be why the workflow was able to complete with the screening options added to the make.contigs command. Screening at the make.contigs step greatly reduces the size of the output files throughout the workflow.
Kindly,
Sarah