# shhh.flows output is very small! What's wrong? **URL:** \ **Category:** Commands in mothur\ **Created:** [March 20, 2014, 9:47am UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760 "2014-03-20T09:47:56Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![Joanito](https://avatars.discourse-cdn.com/v4/letter/j/e47774/32.png) [@Joanito](https://forum.mothur.org/u/Joanito)\ **Post date:** [March 20, 2014, 9:47am UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/1 "2014-03-20T09:47:56Z")
Hi everybody, I’m sorry if this might not be a smart question… I’m having troubles with running the shhh.flows command. I’m just using the exact command from the SOP tutorial. It runs quite quickly (only a few hours) and it doesn’t give any error, but then the output …shhh.fasta and …shhh.names are only a few kilobytes which I assume it should not be possible. I have some hundred thousand sequences so they should be much bigger size. Now I realized that also the output of trim.flows, the …flow.files is only 1kb, while the individual flow files of the different samples are around 30Mb each. But I see from my office mate who ran some samples from the same plates (and have more or less the same amount of sequences) that his individual flow files are about 100Mb… What could be going wrong? Is perhaps something going wrong in the trim.flows step (although I did not get any error)? The 454 machine was a FLX Titanium… Thanks for your help! Joanito ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [March 20, 2014, 11:14am UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/2 "2014-03-20T11:14:33Z")
Can you post the log file of running trim.flows and shhh.flows? Also, could you run summary.seqs using fasta=whatever.shhh.fasta, name=whatever.shhh.names ? Thanks, Pat ---
**Author:** ![Joanito](https://avatars.discourse-cdn.com/v4/letter/j/e47774/32.png) [@Joanito](https://forum.mothur.org/u/Joanito)\ **Post date:** [March 20, 2014, 12:13pm UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/3 "2014-03-20T12:13:55Z")
Dear Pat, here’s the output of summary seqs on the shhh.fasta: Start End NBases Ambigs Polymer NumSeqs Minimum: 1 266 266 0 3 1 2.5%-tile: 1 289 289 0 4 131 25%-tile: 1 299 299 0 5 1307 Median: 1 303 303 0 5 2613 75%-tile: 1 308 308 0 6 3919 97.5%-tile: 1 321 321 0 6 5094 Maximum: 1 347 347 0 9 5224 Mean: 1 303.892 303.892 0 5.07714 # of Seqs: 5224 And this is for the shhh.names: [WARNING]: We found more than 25% of the bases in sequence 3E4NOS02I60VG to be a mbiguous. Mothur is not setup to process protein sequences. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 321414 321414 304273 237 1 2.5%-tile: 1 321414 321414 304273 237 1 25%-tile: 1 321414 321414 304273 237 1 Median: 1 321414 321414 304273 237 1 75%-tile: 1 321414 321414 304273 237 1 97.5%-tile: 1 321414 321414 304273 237 1 Maximum: 1 321414 321414 304273 237 1 Mean: 1 321414 321414 304273 237 # of Seqs: 1 Talking to my colleague again, he told me that sometime ago he had problems with shhh.flows too, and you suggested him to use minflows=200 and maxflows=800 in the trim.seqs step, because of the titanium flx platform. Does this make sense? My data is separated into 5 different sff files, each of which contains many samples but only a few are mine. What I’ve posted here is the output of only one of those 5, for which I’ve now got to the shhh.flows step. I was running the trim.seqs step on those different datasets running mothur in parallel in different terminals. Might have this been the problem and I need to let it go alone for each dataset? (I’ve now seen in the shhh.flows logfile, posted below, that running out of memory might have been the problem). Also, would you suggest a way to merge the data, should I do it at the beginning, or is it best to merge the fasta files after I’ve denoised each separately? (I don’t know if this would be a problem, but some of my samples coming from different runs have the same barcodes) Sorry for so many questions, I’m very new to mothur. Thanks a lot for your kind help, Joanito Here’s the log for the trim.flows and shhh.flows (I hope these are the correct ones. They should be… but I was not aware of the production of log files in the mothur folder and now I’ve lost track of which is which, as I’ve run quite many commands by now. Is there an easy way to understand which log belongs to which file?): mothur \> trim.flows(flow=C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.flow, oligos=C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.oligos, pdiffs=2, bdiffs=1, processors=2) Using 2 processors. 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 240000 250000 260000 270000 280000 290000 300000 310000 320000 330000 340000 350000 360000 370000 380000 390000 400000 410000 420000 430000 440000 450000 460000 470000 480000 490000 500000 510000 520000 530000 540000 550000 560000 570000 580000 590000 600000 610000 620000 630000 640000 650000 660000 670000 680000 690000 700000 710000 718211 Output File Names: C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.trim.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.scrap.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120525-01.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120528-05.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.FungusGardenJOA120528-02.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.FungusGardenJOA120604-03.flow C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.flow.files mothur \> shhh.flows(file=C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.flow.files, processors=2) Unable to open C:\mothur\lookupFiles\LookUp\_Titanium.pat. Trying mothur’s executable location C:\mothur\LookUp\_Titanium.pat Using 2 processors. > > > > > Processing C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120525-01.flow (file 1 of 4) \<\<\<\<\< > > > > > Reading flowgrams… > > > > > Identifying unique flowgrams… > > > > > Calculating distances between flowgrams… Total time: 8 23.478 Clustering flowgrams… \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*# **#** # **#** # **#** # **#** # **#** # Reading matrix: ||||||||||||||||||||||||||||||||||||||||||||||||||| * * * Denoising flowgrams… iter maxDelta nLL cycletime 1 284 -62305.6 10 9.589 2 52.0664 4.48308e+006 2 2.793 3 14.2841 4.87232e+006 2 1.607 4 9.74583 5.01151e+006 1 1.497 5 6.97987 5.04193e+006 2 1.482 6 5.23753 5.04795e+006 1 1.467 7 4.0277 5.04009e+006 2 1.45 8 3.15855 5.04954e+006 1 1.436 9 2.50732 5.05754e+006 2 1.45 10 2.02621 5.05942e+006 1 1.404 11 1.71638 5.06783e+006 2 1.404 12 1.59886 5.07636e+006 1 1.389 13 1.4908 5.0744e+006 1 1.373 14 1.39142 5.07298e+006 2 1.404 15 1.30029 5.07958e+006 1 1.372 16 1.2152 5.08187e+006 1 1.373 17 1.13855 5.07907e+006 2 1.357 18 1.12019 5.08575e+006 1 1.388 19 0.995632 5.08127e+006 2 1.373 20 0.936058 5.08863e+006 1 1.373 21 0.880666 5.08904e+006 1 1.357 22 0.829354 5.08626e+006 2 1.357 23 0.781708 5.09417e+006 1 1.342 … 982 0.000231316 5.10375e+006 1 1.279 983 0.00022947 5.10375e+006 2 1.28 984 0.000227638 5.10375e+006 1 1.279 985 0.000225821 5.10375e+006 1 1.279 986 0.000224019 5.10375e+006 2 1.279 987 0.000222231 5.10375e+006 1 1.28 988 0.000220457 5.10375e+006 1 1.279 989 0.000218698 5.10375e+006 1 1.279 990 0.000216953 5.10375e+006 2 1.279 991 0.000215221 5.10375e+006 1 1.279 992 0.000213504 5.10375e+006 1 1.28 993 0.0002118 5.10375e+006 2 1.279 994 0.000210109 5.10375e+006 1 1.279 995 0.000208432 5.10375e+006 1 1.279 996 0.000206769 5.10375e+006 1 1.28 997 0.000205119 5.10375e+006 2 1.279 998 0.000203482 5.10375e+006 1 1.263 999 0.000201858 5.10375e+006 1 1.264 1000 0.000200247 5.10375e+006 1 1.279 Finalizing… Total time to process C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120525-01.flow: 1298 1298.25 > > > > > Processing C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120528-05.flow (file 2 of 4) \<\<\<\<\< > > > > > Reading flowgrams… > > > > > Identifying unique flowgrams… > > > > > Calculating distances between flowgrams… Total time: 19 32.979 Clustering flowgrams… \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*# **#** # **#** # **#** # **#** # **#** # Reading matrix: ||||||||||||||||||||||||||||||||||||||||||||||||||| * * * Denoising flowgrams… iter maxDelta nLL cycletime 1 137 -44429.8 16 16.537 2 33.0198 4.61613e+006 2 2.418 3 13.8798 4.68538e+006 3 2.075 4 8.71993 4.69671e+006 2 2.012 5 5.92521 4.69381e+006 2 2.028 6 4.21728 4.69955e+006 2 1.997 7 3.18928 4.69956e+006 2 1.997 8 2.91975 4.69749e+006 2 1.981 9 2.65103 4.69121e+006 2 1.997 10 2.43759 4.69721e+006 2 1.981 11 2.43159 4.69919e+006 2 1.981 12 2.34968 4.70156e+006 1 1.981 13 2.28325 4.6951e+006 2 1.982 14 2.20573 4.69041e+006 2 1.981 15 2.11976 4.69123e+006 2 1.965 16 2.02789 4.69171e+006 2 1.982 17 1.93233 4.69788e+006 2 1.965 18 1.835 4.69637e+006 2 1.966 19 1.73755 4.69302e+006 2 1.981 20 1.64117 4.69304e+006 2 1.981 21 1.54699 4.69129e+006 2 1.966 22 1.4557 4.69116e+006 2 1.981 23 1.3679 4.69576e+006 2 1.966 24 1.28402 4.69251e+006 2 1.965 25 1.20422 4.69149e+006 2 1.966 26 1.12861 4.68333e+006 2 1.966 27 1.05727 4.6969e+006 2 1.965 28 1.02966 4.69356e+006 2 1.966 29 1.01437 4.69372e+006 2 1.965 30 1.00755 4.69125e+006 2 1.966 31 0.994544 4.69001e+006 2 1.966 32 0.977814 4.68886e+006 2 1.965 33 0.95652 4.69412e+006 2 1.966 34 0.93144 4.68987e+006 2 1.95 35 0.903169 4.69104e+006 2 1.965 36 0.87229 4.68805e+006 2 1.966 37 0.839367 4.69096e+006 2 1.966 38 0.820901 4.69681e+006 2 1.965 39 0.866073 4.69184e+006 2 1.95 40 0.909824 4.69302e+006 2 1.966 41 0.951587 4.69297e+006 2 1.95 42 0.990804 4.69666e+006 2 1.981 43 1.02694 4.69429e+006 1 1.95 44 1.0595 4.6943e+006 2 1.966 45 1.08805 4.69419e+006 2 1.95 46 1.11223 4.6969e+006 2 1.965 47 1.13176 4.69928e+006 2 1.95 48 1.14645 4.69078e+006 2 1.966 49 1.15621 4.69945e+006 2 1.95 50 1.16103 4.69945e+006 2 1.966 51 1.16101 4.69946e+006 2 1.965 52 1.15632 4.69093e+006 2 1.95 53 1.14718 4.6917e+006 2 1.966 54 1.13392 4.69156e+006 2 1.95 55 1.11686 4.6966e+006 2 1.95 56 1.0964 4.69672e+006 2 1.965 57 1.07292 4.69225e+006 2 1.95 58 1.04685 4.69363e+006 2 1.95 59 1.01857 4.69647e+006 2 1.966 60 0.988499 4.69466e+006 2 1.95 61 0.956999 4.69466e+006 2 1.95 62 0.924428 4.69103e+006 2 1.965 63 0.891113 4.69226e+006 2 1.95 64 0.85735 4.69226e+006 2 1.95 65 0.823406 4.69227e+006 2 1.966 66 0.789516 4.69668e+006 2 1.966 67 0.755885 4.69668e+006 1 1.95 68 0.72269 4.69516e+006 2 1.965 69 0.69008 4.69516e+006 2 1.95 70 0.658178 4.70216e+006 2 1.95 71 0.627085 4.69585e+006 2 1.95 72 0.596882 4.69586e+006 2 1.95 73 0.56763 4.70151e+006 2 1.966 74 0.539374 4.69615e+006 2 1.965 75 0.512145 4.69616e+006 2 1.95 76 0.485962 4.6979e+006 2 1.95 77 0.460833 4.69791e+006 2 1.935 78 0.436756 4.69791e+006 2 1.95 79 0.413725 4.69791e+006 2 1.95 80 0.391724 4.69792e+006 2 1.95 81 0.370734 4.69792e+006 2 1.95 82 0.35073 4.69792e+006 2 1.95 83 0.331687 4.69793e+006 2 1.966 84 0.313576 4.69793e+006 2 1.95 85 0.296364 4.69793e+006 2 1.95 86 0.28002 4.70227e+006 2 1.95 87 0.264511 4.70445e+006 2 1.966 88 0.249804 4.70461e+006 2 1.965 89 0.235864 4.70461e+006 1 1.966 90 0.222659 4.70496e+006 2 1.965 91 0.210157 4.69679e+006 2 1.95 92 0.198324 4.70396e+006 2 1.966 93 0.187129 4.70615e+006 2 1.966 94 0.176542 4.70762e+006 2 1.95 95 0.166532 4.70977e+006 2 1.965 96 0.157072 4.70977e+006 2 1.95 97 0.148134 4.70977e+006 2 1.981 98 0.13969 4.70977e+006 2 1.95 99 0.131715 4.70977e+006 2 1.95 100 0.124186 4.70977e+006 2 1.95 101 0.120387 4.71025e+006 2 1.95 102 0.118533 4.71025e+006 2 1.951 103 0.116739 4.7062e+006 2 1.965 104 0.114794 4.7062e+006 2 1.95 105 0.112829 4.7062e+006 2 1.95 106 0.110836 4.7062e+006 2 1.95 107 0.10882 4.7062e+006 2 1.935 108 0.106783 4.7062e+006 2 1.935 109 0.10473 4.7062e+006 2 1.95 110 0.102665 4.7062e+006 2 1.95 111 0.100592 4.7062e+006 1 1.95 112 0.0986145 4.71042e+006 2 1.95 113 0.0966191 4.70709e+006 2 1.934 114 0.0945235 4.70709e+006 2 1.95 115 0.0924466 4.70662e+006 2 1.95 116 0.0904771 4.70753e+006 2 1.95 117 0.0884008 4.70753e+006 2 1.95 118 0.0863516 4.70829e+006 2 1.95 119 0.084317 4.70829e+006 2 1.95 120 0.0822992 4.70829e+006 2 1.95 121 0.0803 4.70932e+006 2 1.95 122 0.0783214 4.70932e+006 2 1.95 123 0.0763652 4.70932e+006 2 1.95 124 0.0744328 4.70932e+006 2 1.95 125 0.0725257 4.70932e+006 2 1.95 126 0.0706451 4.70932e+006 2 1.95 127 0.0687923 4.70932e+006 2 1.95 128 0.099824 4.70191e+006 2 1.95 129 0.0651738 4.70191e+006 2 1.95 130 0.0634098 4.70191e+006 2 1.95 131 0.0616769 4.70191e+006 1 1.95 132 0.0599758 4.70191e+006 2 1.95 133 0.0583069 4.70191e+006 2 1.935 134 0.0566706 4.70191e+006 2 1.965 135 0.0550671 4.70191e+006 2 1.95 136 0.0534968 4.70192e+006 2 1.95 137 0.0519598 4.70192e+006 2 1.95 138 0.0504561 4.70192e+006 2 1.935 139 0.0489858 4.70192e+006 2 1.95 140 0.0475488 4.70192e+006 2 1.95 141 0.046145 4.70192e+006 2 1.95 142 0.0447743 4.70192e+006 2 1.95 143 0.0434364 4.70192e+006 2 1.934 144 0.0421311 4.70192e+006 2 1.95 145 0.0408581 4.70192e+006 2 1.95 146 0.039617 4.70192e+006 2 1.95 147 0.0384076 4.70192e+006 2 1.95 148 0.0372294 4.70192e+006 2 1.95 149 0.036082 4.70192e+006 2 1.95 150 0.034965 4.70192e+006 1 1.935 151 0.0339781 4.70222e+006 2 1.95 152 0.0329 4.70222e+006 2 1.95 153 0.0318688 4.70222e+006 2 1.95 154 0.0996921 4.7064e+006 2 1.95 155 0.0298914 4.7064e+006 2 1.965 156 0.028944 4.7064e+006 2 1.95 157 0.0280235 4.70958e+006 2 1.935 158 0.0271294 4.70958e+006 2 1.95 159 0.026261 4.70958e+006 2 1.95 160 0.0254178 4.70958e+006 2 1.95 161 0.0245992 4.70958e+006 2 1.95 162 0.0238048 4.70958e+006 2 1.95 163 0.0230339 4.70958e+006 2 1.95 164 0.022286 4.71348e+006 2 1.95 165 0.0215606 4.71348e+006 2 1.966 166 0.020857 4.71348e+006 2 1.934 167 0.0201748 4.71348e+006 2 1.966 168 0.0195134 4.71348e+006 2 1.934 169 0.0188723 4.71348e+006 2 1.95 170 0.0182509 4.71348e+006 1 1.95 171 0.0176488 4.71348e+006 2 1.95 172 0.0170654 4.71348e+006 2 1.95 173 0.0165002 4.71348e+006 2 1.95 174 0.0159528 4.71349e+006 2 1.95 175 0.0154225 4.71349e+006 2 1.95 176 0.014909 4.71349e+006 2 1.95 177 0.0144119 4.71349e+006 2 1.935 178 0.0139305 4.71349e+006 2 1.95 179 0.0134645 4.71349e+006 2 1.95 180 0.0990421 4.70882e+006 2 1.934 181 0.0125769 4.70882e+006 2 1.95 182 0.0121544 4.70882e+006 2 1.95 183 0.0117455 4.70882e+006 2 1.95 184 0.01135 4.70882e+006 2 1.95 185 0.0109672 4.70882e+006 2 1.935 186 0.010597 4.70882e+006 2 1.95 187 0.0102388 4.70882e+006 2 1.935 188 0.00989239 4.70882e+006 2 1.95 189 0.00955733 4.70882e+006 1 1.95 190 0.00923329 4.70882e+006 2 1.95 191 0.00891993 4.70882e+006 2 1.95 192 0.00861692 4.70882e+006 2 1.934 193 0.00832393 4.70882e+006 2 1.95 194 0.00804066 4.70882e+006 2 1.95 195 0.0077668 4.70882e+006 2 1.95 196 0.00750205 4.70882e+006 2 1.95 197 0.00724612 4.70882e+006 2 1.95 198 0.00699873 4.70882e+006 2 1.95 199 0.00675962 4.70882e+006 2 1.95 200 0.00652851 4.70882e+006 2 1.935 201 0.00630515 4.70882e+006 2 1.95 202 0.00608929 4.70882e+006 2 1.95 203 0.00588069 4.70882e+006 2 1.934 204 0.00567911 4.70882e+006 2 1.95 205 0.00548433 4.70882e+006 2 1.95 206 0.00529612 4.70882e+006 2 1.935 207 0.00511426 4.70882e+006 2 1.965 208 0.00493856 4.70882e+006 1 1.935 209 0.00476881 4.70882e+006 2 1.935 210 0.00460482 4.70882e+006 2 1.95 211 0.00444638 4.70882e+006 2 1.95 212 0.00429333 4.70882e+006 2 1.934 213 0.00414548 4.70882e+006 2 1.95 214 0.00400266 4.70882e+006 2 1.95 215 0.00386471 4.70882e+006 2 1.935 216 0.00373146 4.70882e+006 2 1.95 217 0.00360275 4.70882e+006 2 1.95 218 0.00347843 4.70882e+006 2 1.95 219 0.00335837 4.70882e+006 2 1.934 220 0.0032424 4.70882e+006 2 1.95 221 0.00313041 4.70882e+006 2 1.95 222 0.00302224 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1.95 627 2.16853e-006 4.70882e+006 2 1.95 628 2.14619e-006 4.70882e+006 2 1.95 629 2.12407e-006 4.70882e+006 2 1.934 630 2.10218e-006 4.70882e+006 2 1.95 631 2.08052e-006 4.70882e+006 2 1.95 632 2.05908e-006 4.70882e+006 2 1.95 633 2.03787e-006 4.70882e+006 2 1.95 634 2.01687e-006 4.70882e+006 2 1.95 635 1.99609e-006 4.70882e+006 2 1.95 636 1.97552e-006 4.70882e+006 2 1.935 637 1.95516e-006 4.70882e+006 1 1.95 638 1.93502e-006 4.70882e+006 2 1.95 639 1.91508e-006 4.70882e+006 2 1.95 640 1.89534e-006 4.70882e+006 2 1.95 641 1.87581e-006 4.70882e+006 2 1.934 642 1.85649e-006 4.70882e+006 2 1.934 643 1.83736e-006 4.70882e+006 2 1.95 644 1.81842e-006 4.70882e+006 2 1.95 645 1.79969e-006 4.70882e+006 2 1.95 646 1.78114e-006 4.70882e+006 2 1.935 647 1.76279e-006 4.70882e+006 2 1.95 648 1.74463e-006 4.70882e+006 2 1.95 649 1.72665e-006 4.70882e+006 2 1.95 650 1.70886e-006 4.70882e+006 2 1.934 651 1.69125e-006 4.70882e+006 2 1.95 652 1.67383e-006 4.70882e+006 2 1.935 653 1.65658e-006 4.70882e+006 2 1.935 654 1.63951e-006 4.70882e+006 2 1.95 655 1.62262e-006 4.70882e+006 2 1.95 656 1.6059e-006 4.70882e+006 1 1.95 657 1.58935e-006 4.70882e+006 2 1.95 658 1.57298e-006 4.70882e+006 2 1.95 659 1.55677e-006 4.70882e+006 2 1.965 660 1.54073e-006 4.70882e+006 2 1.935 661 1.52485e-006 4.70882e+006 2 1.934 662 1.50914e-006 4.70882e+006 2 1.95 663 1.49359e-006 4.70882e+006 2 1.95 664 1.4782e-006 4.70882e+006 2 1.95 665 1.46297e-006 4.70882e+006 2 1.95 666 1.4479e-006 4.70882e+006 2 1.95 667 1.43298e-006 4.70882e+006 2 1.934 668 1.41822e-006 4.70882e+006 2 1.951 669 1.4036e-006 4.70882e+006 2 1.95 670 1.38914e-006 4.70882e+006 2 1.95 671 1.37483e-006 4.70882e+006 2 1.95 672 1.36066e-006 4.70882e+006 2 1.95 673 1.34664e-006 4.70882e+006 2 1.95 674 1.33277e-006 4.70882e+006 2 1.934 675 1.31904e-006 4.70882e+006 1 1.95 676 1.30545e-006 4.70882e+006 2 1.95 677 1.292e-006 4.70882e+006 2 1.95 678 1.27868e-006 4.70882e+006 2 1.935 679 1.26551e-006 4.70882e+006 2 1.95 680 1.25247e-006 4.70882e+006 2 1.95 681 1.23957e-006 4.70882e+006 2 1.965 682 1.22679e-006 4.70882e+006 2 1.95 683 1.21415e-006 4.70882e+006 2 1.935 684 1.20165e-006 4.70882e+006 2 1.935 685 1.18926e-006 4.70882e+006 2 1.95 686 1.17701e-006 4.70882e+006 2 1.95 687 1.16488e-006 4.70882e+006 2 1.95 688 1.15288e-006 4.70882e+006 2 1.95 689 1.141e-006 4.70882e+006 2 1.95 690 1.12925e-006 4.70882e+006 2 1.965 691 1.11761e-006 4.70882e+006 2 1.95 692 1.1061e-006 4.70882e+006 2 1.935 693 1.0947e-006 4.70882e+006 2 1.95 694 1.08342e-006 4.70882e+006 2 1.95 695 1.07226e-006 4.70882e+006 1 1.95 696 1.06121e-006 4.70882e+006 2 1.934 697 1.05028e-006 4.70882e+006 2 1.95 698 1.03946e-006 4.70882e+006 2 1.95 699 1.02875e-006 4.70882e+006 2 1.95 700 1.01815e-006 4.70882e+006 2 1.935 701 1.00766e-006 4.70882e+006 2 1.95 702 9.97279e-007 4.70882e+006 2 1.95 Finalizing… Total time to process C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.S.amabilisJOA120528-05.flow: 1386 1386.19 > > > > > Processing C:\Users\xtr552\Desktop\MothurAnalysis\ThirdRun\H3E4NOS02.FungusGardenJOA120528-02.flow (file 3 of 4) \<\<\<\<\< > > > > > Reading flowgrams… > > > > > Identifying unique flowgrams… > > > > > Calculating distances between flowgrams… Total time: 30 67.19 Clustering flowgrams… \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*# **#** # **#** # **#** # **#** # **#** # Reading matrix: ||||||||||||||||||||||||||||||||||||||||||||||||||| * * * [ERROR]: std::bad\_alloc has occurred in the ShhherCommand class function driver. This error indicates your computer is running out of memory. This is most commonly caused by trying to process a dataset too large, using multiple processors, or a file format issue. If you are running our 32bit version, your memory usage is limited to 4G. If you have more than 4G of RAM and are running a 64bit OS, using our 64bit version may resolve your issue. If you are using multiple processors, try running the command with processors=1, the more processors you use the more memory is required. Also, you may be able to reduce the size of your dataset by using the commands outlined in the Schloss SOP, [http://www.mothur.org/wiki/Schloss\_SOP](http://www.mothur.org/wiki/Schloss_SOP). If you are uable to resolve the issue, please contact Pat Schloss at [mothur.bugs@gmail.com](mailto:mothur.bugs@gmail.com), and be sure to include the mothur.logFile with your inquiry. ---
**Author:** ![Joanito](https://avatars.discourse-cdn.com/v4/letter/j/e47774/32.png) [@Joanito](https://forum.mothur.org/u/Joanito)\ **Post date:** [March 25, 2014, 2:01pm UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/4 "2014-03-25T14:01:36Z")
Hi everybody, I’ve now got to repeat the analysis using sff multiple in order to merge all the data that is coming from 5 different runs (I have only a few samples that are mine in each of the file and some of the barcodes overlap between runs). So I have prepared 5 different .oligos files to map the correct samples in the sff files. It all works, but at the end of all the process I get only 8 unique sequences across all my 33 samples! This is impossible as I’m just redoing the analysis with Mothur after I’ve analyzed everything with Qiime and I know there are about 1000 OTUs in my dataset. I’ve also tried to redo every step singularly and I can see that the output of shhh.flows is very small, the shhh.fasta files are just a few kilobytes and contain 4 or 5 senquences per run. I’m just using the exact commands I find in the SOP tutorial and I get no error message. Now I’m using a very powerful pc with 4 cores so I know it’s not a problem of memory runout like I had before. What could be going wrong? I’m sorry if this sounds basic, I hope you can help me. Thanks! Joanito ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [March 31, 2014, 3:33pm UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/5 "2014-03-31T15:33:35Z")
What version of mothur are you running? Can you tell where the number of uniques is dropping? Is mothur scrapping your flows after the trim.flows command or are the sequences being put into the names file in the shhh.flows command? ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [April 3, 2014, 11:01am UTC](https://forum.mothur.org/t/shhh-flows-output-is-very-small-whats-wrong/1760/6 "2014-04-03T11:01:01Z")
Can you run this instead… summary.seqs(fasta=shhh.fasta, names=shhh.names) Instead of shhh.fasta and shhh.names, use your specific files. Pat