Hi,
I am currently trying to use mothur to analyse 454 data that was generated as follows: I had one main sample which was PCR amplified with a non-barcoded primer pair, and 20 others samples that were PCR amplified individually with different barcoded primers were mixed into this non-barcoded sample. So in my oligos file I have the forward primer and my 20 barcodes. I want to use trim.flows to separate out the flowgrams so that I have files for the main non-barcoded sample and all the barcoded ones, but when I do this, I have the feeling that the non-barcoded sequences are being scraped. If I look in .flow.files, I have a list of files for the 20 barcoded samples, but not for the non-barcoded one. And the .trim.flow file that is produced that does not contain a barcode prefix seems too small in size to be the main non-barcoded sample. So essentially I would like to check what happens to sequences that do not contain barcodes when trim.flows is run with barcodes in the oligos file.
I hope that made sense and thanks a lot,
Emily
So if your sample didn’t have a barcode, it will go to the scrap.fasta file. That file will also have things that are a couple of bases off of the samples that did have the barcodes. But your samples without a barcode should start with the primer. Can you try running scrap.fasta through trim.flows again but use an oligos file that doesn’t have any barcodes - just the primer?
That’s a good idea! Thanks for confirming what happens to the sample without a barcode and the suggestion. I will have a go.
Thanks a lot,
Emily
If I try running:
trim.flows(flow=IHV8ERT01.scrap.flow, oligos=IHV8ERT01primer.oligos)
I get the error message:
Error in reading your flowfile, at position -1. Blank name.
Do you know what this error could mean? The first and following line of my scrap.flow file look like this:
450
IHV8ERT01EFD8V|lb 141 1.05 0.04 0.99 0.05 0.00 1.00 0.01 1.02 0.03 1.02 0.03 2.56 0.07 0.00 0.26 0.00 0.04 0.00 0.00 1.06
I wondered if the error could be something to do with the names for each sequence in the first column of the file, and tried changing them from e.g. IHV8ERT01EFD8V|lb to IHV8ERT01EFD8V (i.e. as they were in my initial .flow file). I still get the same error though. Any help would be much appreciated.
Thanks!
Emily
That error message usually occurs when mothur is trying to read past the end of the file. Can you try adding a blank line at the end of the *.scrap.flow file?
Thanks for the reply. I added a blank line at the end of my .scrap.flow file but I get the same error message. Mothur does start to write something into a .scrap.scrap.flow (see below). It also did this before I added the blank line, but it seems as though the file is getting written a bit funny - it looks as though the names for the sequences are getting miswritten (e.g. 0.16|lf)?
450
IHV8ERT01EFD8V|lb|l 141 1.05 0.04 0.99 0.05 0.00 1.00 0.01 1.02 0.03 1.02 0.03 2.56 0.07 0.00 0.26 0.00 0.04 0.00 0.00 1.06 0.02 1.80 0.39 0.46 0.03 1.50 0.77 0.04 0.82 0.01 0.00 1.85 0.06 0.01 0.00 0.00 0.02 0.00 1.02 0.03 0.00 1.00 0.03 0.00 2.05 1.03 0.05 1.01 0.03 0.00 1.07 0.02 0.00 1.02 1.03 0.03 2.08 0.06 0.98 0.99 0.06 2.04 0.04 1.09 2.01 2.04 0.03 0.00 2.09 0.99 0.99 0.00 0.00 1.03 0.03 1.04 0.02 2.03 1.96 0.07 1.05 0.02 0.98 0.00 0.00 0.97 0.01 0.00 1.09 1.00 0.99 0.02 1.05 1.00 0.03 1.04 0.01 0.00 1.91 2.55 1.04 0.07 0.04 1.98 0.04 0.96 0.01 0.01 1.91 0.97 0.06 0.00 0.93 0.04 1.87 0.07 1.84 0.06 0.00 0.00 3.25 0.06 1.04 0.04 1.08 0.00 0.02 1.10 1.05 0.97 0.04 0.05 0.98 0.99 0.03 0.00 0.97 2.09 0.03 0.00 1.95 0.00 0.00 0.00 1.02 2.85 0.05 0.01 0.04 0.00 0.00 1.05 1.73 0.07 0.00 0.97 0.04 0.00 0.00 0.00 0.00 0.00 0.00 2.61 0.10 0.00 0.21 0.03 0.03 0.03 0.98 0.93 0.04 0.00 1.76 1.69 0.08 0.02 0.89 0.00 0.06 0.00 0.99 0.00 0.00 0.00 1.94 2.56 0.11 0.00 0.95 0.02 1.71 0.06 0.02 1.84 0.08 0.02 0.88 0.06 0.92 0.05 0.05 1.77 0.06 0.90 1.69 0.05 0.08 0.92 0.89 0.09 0.02 0.04 0.02 0.04 0.88 0.04 0.01 0.06 0.98 1.00 0.97 1.01 0.02 0.04 4.01 0.18 0.03 0.03 1.02 0.26 0.09 0.01 2.57 0.78 0.77 0.82 0.05 1.03 1.16 1.09 0.12 0.04 0.00 2.99 1.04 0.05 0.06 2.15 0.35 0.04 0.34 1.16 0.04 0.01 0.02 1.01 0.00 0.01 0.00 1.04 0.03 0.04 2.11 0.07 0.00 0.00 2.95 0.12 0.00 1.96 1.07 0.09 2.76 0.13 0.05 0.94 1.16 0.13 0.96 0.14 0.96 0.93 0.01 0.14 0.00 0.00 0.02 2.03 1.07 0.10 0.20 0.09 0.00 1.01 0.02 2.10 1.01 0.04 1.02 0.03 2.07 0.09 0.03 1.00 0.95 0.07 1.93 0.12 0.07 0.03 0.98 0.03 2.07 0.04 0.01 0.00 0.94 0.06 0.97 1.97 1.18 0.06 0.00 1.08 0.05 1.04 0.06 1.03 0.04 0.02 0.00 0.98 0.12 0.00 0.08 0.89 2.16 2.98 0.13 0.02 0.91 0.03 0.00 0.03 0.00 1.05 3.01 0.13 0.03 0.98 0.06 0.06 3.91 0.18 0.08 0.09 0.04 0.00 2.17 1.16 0.06 1.01 0.12 0.05 0.96 1.13 1.02 1.00 1.04 0.13 1.18 0.00 0.96 2.73 0.92 0.07 0.00 0.08 0.87 0.00 0.07 2.14 0.07 0.02 0.96 0.10 0.93 0.03 2.07 0.12 0.15 0.92 0.04 0.10 0.86 0.04 0.06 0.05 2.10 0.09 1.11 0.18 1.11 1.97 1.02 0.03 2.24 1.04 0.05 0.00 0.98 0.04 2.03 1.08 1.07 0.03 0.02 0.04 0.01 1.04 0.04 0.13 0.00 0.00 0.00 0.99 0.05 1.11 0.02 0.00 1.13 0.06 1.08 0.00 0.07 2.00 0.13 0.06 0.00 1.93 0.05 1.15 0.09 1.91 0.15 1.00 0.09 0.93
0.09|lf 0 0.07 0.89 0.07 0.02 1.90 1.13 0.10 1.02 1.93 0.11 0.04 1.93 1.08 0.07 0.12 1.05 1.05 1.07 0.17 0.04 3.02 0.08 1.01 0.14 1.08 1.01 0.07 0.15 3.62 0.11 0.79 0.06 0.09 1.68 2.06 0.19 0.03 0.04 0.02 3.89 1.10 1.99 0.10 2.66 1.18 0.09 0.02 0.98 0.89 0.11 0.04 0.99 0.02 0.26 0.07 0.78 0.08 2.18 0.10 1.81 1.39 0.11 0.05 0.02 0.00 1.07 0.04 0.03 1.17 0.07 0.04 0.03 0.00 0.00 2.00 1.97 0.94 0.07 0.03 0.09 0.94 3.14 0.17 0.07 0.06 1.02 1.06 0.06 0.06 0.99 0.14 0.88 1.00 0.15 0.03 0.05 1.01 3.23 0.18 1.43 1.06 1.05 2.93 0.11 0.17 0.09 0.01 0.00 1.09 0.13 0.17 0.00 0.00 0.02 0.00 1.07 1.18 0.07 0.09 2.09 0.09 1.05 2.86 0.13 0.10 0.28 0.08 0.10 0.82 1.07 0.05 1.02 0.24 0.12 0.03 0.92 0.00 0.93 0.16 0.05 1.03 0.04 1.16 0.20 0.04 0.00 0.07 2.06 1.12 0.07 0.11 0.98 0.05 0.01 0.07 2.99 0.18 2.06 0.09 0.14 1.02 2.00 2.11 1.10 0.10 2.04 0.97 2.66 0.14 0.11 0.04 0.01 0.03 0.97 0.05 0.09 2.10 1.04 0.09 1.03 0.26 1.05 0.12 3.07 1.10 1.21 0.17 0.07 0.97 0.12 0.05 0.08 0.11 1.01 1.02 0.05 0.05 1.08 0.26 0.98 0.11 1.09 0.08 3.11 1.10 1.08 0.19 1.11 3.32 1.07 0.11 2.20 2.31 1.11 0.14 0.01 0.04 1.41 0.91 0.77 0.04 0.02 0.02 3.35 0.07 0.06 0.84 0.11 0.07 0.80 1.97 0.14 0.06 0.05 0.01 1.99 0.97 1.09 1.94 1.06 0.11 0.01 2.10 0.10 0.88 0.05 0.20 0.03 0.92 2.90 0.21 0.16 1.01 0.05 0.06 2.10 0.99 3.57 0.19 1.34 0.76 1.42 0.08 1.26 0.74 0.62 0.81 1.00 1.00 1.27 1.50 0.08 0.23 0.04 1.07 0.23 0.03 0.02 0.25 0.86 0.13 0.01 1.07 0.08 1.19 0.05 0.04 1.19 0.11 0.22 0.23 0.28 1.37 0.08 0.01 0.24 0.73 0.07 2.70 0.34 0.34 0.72 0.10 0.04 0.05 1.12 0.11 0.01 1.07 0.18 0.16 1.05 1.08 0.11 0.02 0.05 1.17 0.25 0.19 0.84 0.18 1.12 1.01 0.06 0.06 0.00 0.04 2.31 0.17 2.12 2.21 0.95 1.02 0.33 0.96 1.21 0.10 1.05 0.04 0.12 1.19 0.06 4.25 0.11 0.97 0.53 0.83 0.97 0.35 1.27 0.32 0.09 0.36 0.53 0.21 0.05 0.56 0.12 0.09 0.21 0.10 0.01 0.30 0.09 0.22 0.08 0.30 0.03 0.18 0.27 0.12 0.47 0.22 0.52 0.21 0.09 0.00 0.03 0.70 0.07 0.10 0.29 0.09 0.09 0.00 0.17 0.25 0.10 0.19 0.09 0.08 0.17 0.02 0.00 0.19 0.44 0.05 0.05 0.23 0.09 0.03 0.01 0.01 0.00 0.26 0.22 0.00 0.27 0.10 0.13 0.10 0.16 0.12 0.00 0.11 0.11 0.11 0.08 0.04 0.13 0.10 0.07 0.00 0.12 0.15 0.09 0.09 0.13 0.09 0.07 0.06 0.06 0.00 0.16 0.03 0.00 0.01 0.23 0.03 0.10 0.11 0.03 0.10 0.11 0.09 0.03 0.06 0.14 0.05 0.06
0.16|lf 0 0.06 0.11 0.08 0.06 0.00 0.09 0.09 0.04 0.08 0.04 0.11 0.02 0.04 0.02 0.12 0.01 0.06 0.00 0.00 0.00 0.15 0.07 0.05 0.00 0.00 0.08 0.03 0.16 0.04 0.07 0.07 0.05 0.00 0.05 0.11 0.10 0.06 0.06 0.02 0.08 0.01 0.00 0.08 0.09 0.01 0.03 0.12 0.10 0.05 0.04 0.01 0.00 0.16 0.06 0.01 0.11 0.05 0.07 0.09 0.10 0.07 0.00 0.04 0.12 0.04 0.01 0.05 0.12 0.04 0.02 0.04 0.06 0.01 0.10 0.02 0.00 0.09 0.07 0.12 0.08 0.02 0.02 0.03 0.11 0.03 0.03 0.00 0.12 0.06 0.02 0.00 0.02 0.13 0.07 0.05 0.08 0.07 0.04 0.00 0.02 0.11 0.06 0.04 0.01 0.04 0.00 0.00 0.07 0.03 0.07 0.01 0.01 0.04 0.10 0.00 0.04 0.01 0.00 0.00 0.09 0.08 0.02 0.01 0.00 0.03 0.00 0.09 0.05 0.04 0.06 0.05 0.00 0.04 0.09 0.01 0.07 0.00 0.05 0.02 0.03 0.02 0.06 0.01 0.06 0.02 0.03 0.00 0.06 0.03 0.00 0.02 0.09 0.02 0.03 0.09 0.05 0.03 0.06 0.06 0.01 0.03 0.10 0.01 0.04 0.05 0.01 0.05 0.07 0.02 0.12 0.04 0.04 0.03 0.02 0.00 0.09 0.04 0.07 0.03 0.02 0.02 0.04 0.05 0.01 0.05 0.00 0.06 0.06 0.02 0.00 0.01 0.05 0.03 0.00 0.06 0.02 0.04 0.00 0.01 0.10 0.03 0.01 0.00 0.02 0.00 0.01 0.05 0.03 0.07 0.02 0.05 0.02 0.08 0.00 0.02 0.00 0.00 0.00 0.05 0.07 0.01 0.02 0.00 0.04 0.00 0.07 0.03 0.02 0.04 0.03 0.00 0.03 0.06 0.04 0.04 0.04 0.01 0.03 0.00 0.00 0.06 0.04 0.04 0.03 0.03 0.01 0.02 0.02 0.03 0.00 0.06 0.04 0.01 0.02 0.01 0.03 0.05 0.05 0.02 0.00 0.01 0.07 0.02 0.00 0.02 0.07 0.00 0.02 0.00 0.03 0.02 0.04 0.01 0.03 0.02 0.03 0.02 0.02 0.02 0.04 0.01 0.00 0.02 0.12 0.00 0.02 0.04 0.01 0.01 0.04 0.02 0.01 0.02 0.04 0.00 0.05 0.02 0.02 0.04 0.03 0.03 0.03 0.01 0.04 0.00 0.00 0.03 0.04 0.02 0.01 0.05 0.02 0.01 0.03 0.02 0.00 0.08 0.04 0.00 0.04 0.02 0.01 0.06 0.05 0.04 0.00 0.01 0.06 0.02 0.00 0.02 0.04 0.01 0.01 0.00 0.03 0.04 0.01 0.03 0.05 0.01 0.00 0.03 0.01 0.00 0.05 0.00 0.00 0.01 0.07 0.00 0.03 0.05 0.01 0.02 0.05 0.04 0.00 0.04 0.06 0.02 0.03 0.02 0.03 0.04 0.02 0.02 0.01 0.03 0.02 0.04 0.01 0.00 0.03 0.06 0.01 0.05 0.02 0.00 0.03 0.05 0.00 0.04 0.00 0.06 0.04 0.02 0.00 0.02 0.05 0.00 0.01 0.05 0.01 0.02 0.00 0.02 0.05 0.02 0.01 0.00 0.03 0.00 0.00 0.02 0.01 0.05 0.00 0.00 0.02 0.08 0.00 0.03 0.00 0.00 0.00 0.03 0.04 0.01 0.00 0.00 0.03 0.00 0.05 0.04 0.01 0.02 0.04 0.00 0.01 0.04 0.05 0.02 0.03 0.01 0.02 0.00 0.00 0.03 0.03 0.02 0.01 0.03 0.04 0.00 0.03 0.00 0.00 0.04 0.02
0.00|lf 0 0.02 0.00 0.02 0.04 0.02 0.02 0.00 0.01 0.05 0.01 0.00 0.02 0.03 0.01 0.00 0.00 0.04 0.00 0.00 0.03 0.00 0.03 0.00 0.00 0.01 0.04 0.00 0.03 0.00 0.00 0.00 0.06 0.03 0.00 0.00 0.00 0.02 0.00 0.06 0.02 0.01 0.01 0.02 0.00 0.00 0.05 0.02 0.03 0.00 0.02 0.03 0.02 0.02 0.04 0.01 0.02 0.01 0.02 0.00 0.05 0.00 0.02 0.03 0.03 0.00 0.01 0.04 0.01 0.00 0.03 0.02 0.00 0.00 0.05 0.00 0.03 0.02 0.00 0.05 0.01 0.01 0.02 0.02 0.01 0.03 0.00 0.00 0.03 0.02 0.00 0.02 0.01 0.01 0.04 0.02 0.00 0.01 0.00 0.02 0.05 0.03 0.00 0.00 0.03 0.01 0.02 0.02 0.01 0.02 0.00 0.00 0.05 0.00 0.02 0.00 0.03 0.01 0.04 0.01 0.03 0.01 0.01 0.00 0.00 0.00 0.04 0.01 0.00 0.03 0.02 0.00 0.01 0.03 0.00 0.00 0.03 0.01 0.00 0.01 0.05 0.00 0.02 0.01 0.00 0.05 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.00 0.05 0.00 0.00 0.03 0.03 0.01 0.02 0.00 0.00 0.00 0.04 0.02 0.00 0.00 0.00 0.03 0.00 0.03 0.02 0.01 0.02 0.01 0.00 0.00 0.04 0.05 0.03 0.00 0.02 0.00 0.03 0.00 0.00 0.03 0.03 0.00 0.03 0.01 0.00 0.02 0.01 0.00 0.01 0.00 0.03 0.04 0.01 0.00 0.00 0.03 0.00 0.01 0.04 0.01 0.01 0.00 0.00 0.04 0.01 0.00 0.02 0.00 0.01 0.01 0.00 0.04 0.01 0.00 0.01 0.03 0.02 0.00 0.02 0.00 0.00 0.05 0.03 0.00 0.02 0.00 0.01 0.02 0.02 0.02 0.00 0.00 0.05 0.00 0.00 0.01 0.03 0.00 0.02 0.00 0.02 0.01 0.01 0.01 0.04 0.03 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.02 0.01 0.00 0.01 0.04 0.00 0.00 0.02 0.00 0.00 0.00 0.03 0.01 0.01 0.01 0.00 0.02 0.00 0.00 0.00 0.03 0.00 0.00 0.01 0.00 0.03 0.00 0.00 0.02 0.04 0.00 0.02 0.00 0.00 0.00 0.04 0.01 0.00 0.00 0.00 0.02 0.00 0.03 0.04 0.01 0.00 0.01 0.00 0.00 0.03 0.01 0.01 0.03 0.00 0.02 0.00 0.00 0.04 0.02 0.00 0.01 0.02 0.01 0.00 0.02 0.00 0.00 0.03 0.04 0.00 0.02 0.00 0.01 0.02 0.00 0.01 0.00 0.01 0.05 0.00 0.00 0.01 0.03 0.02 0.01 0.00 0.04 0.00 0.02 0.00 0.00 0.02 0.01 0.00 0.02 0.00 0.00 0.02 0.02 0.00 0.00 0.00 0.03 0.01 0.01 0.00 0.00 0.01 0.02 0.01 0.02 0.02 0.00 0.00 0.00 0.05 0.00 0.02 0.00 0.02 0.01 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.01 0.01 0.00 0.00 0.05 0.00 0.00 0.03 0.00 0.00 0.00 0.03 0.00 0.01 0.01 0.00 0.01 0.00 0.00 0.02 0.00 0.01 0.00 0.00 0.02 0.02 0.00 0.00 0.03 0.00 0.00 0.02 0.00 0.00 0.00 0.04 0.00 0.01 0.04 0.05 0.02 0.03 0.01 0.02 0.00 0.00 0.03 0.03 0.02 0.01 0.03 0.04 0.00 0.03 0.00 0.00 0.04 0.02
|lf 0 0.02 0.00 0.02 0.04 0.02 0.02 0.00 0.01 0.05 0.01 0.00 0.02 0.03 0.01 0.00 0.00 0.04 0.00 0.00 0.03 0.00 0.03 0.00 0.00 0.01 0.04 0.00 0.03 0.00 0.00 0.00 0.06 0.03 0.00 0.00 0.00 0.02 0.00 0.06 0.02 0.01 0.01 0.02 0.00 0.00 0.05 0.02 0.03 0.00 0.02 0.03 0.02 0.02 0.04 0.01 0.02 0.01 0.02 0.00 0.05 0.00 0.02 0.03 0.03 0.00 0.01 0.04 0.01 0.00 0.03 0.02 0.00 0.00 0.05 0.00 0.03 0.02 0.00 0.05 0.01 0.01 0.02 0.02 0.01 0.03 0.00 0.00 0.03 0.02 0.00 0.02 0.01 0.01 0.04 0.02 0.00 0.01 0.00 0.02 0.05 0.03 0.00 0.00 0.03 0.01 0.02 0.02 0.01 0.02 0.00 0.00 0.05 0.00 0.02 0.00 0.03 0.01 0.04 0.01 0.03 0.01 0.01 0.00 0.00 0.00 0.04 0.01 0.00 0.03 0.02 0.00 0.01 0.03 0.00 0.00 0.03 0.01 0.00 0.01 0.05 0.00 0.02 0.01 0.00 0.05 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.00 0.05 0.00 0.00 0.03 0.03 0.01 0.02 0.00 0.00 0.00 0.04 0.02 0.00 0.00 0.00 0.03 0.00 0.03 0.02 0.01 0.02 0.01 0.00 0.00 0.04 0.05 0.03 0.00 0.02 0.00 0.03 0.00 0.00 0.03 0.03 0.00 0.03 0.01 0.00 0.02 0.01 0.00 0.01 0.00 0.03 0.04 0.01 0.00 0.00 0.03 0.00 0.01 0.04 0.01 0.01 0.00 0.00 0.04 0.01 0.00 0.02 0.00 0.01 0.01 0.00
Could you send your logfile, IHV8ERT01.scrap.flow and IHV8ERT01primer.oligos to mothur.bugs@gmail.com so I can track down the issue for you?
Hi Emily,
Thank you for sending your files. I was able to track down the source of the problem. Mothur was printing the number of trimmed flows in the scrap flow file instead of the original number of flows. This was causing a read error when the scrap file was read back in. Here’s an example of what I mean. Your file starts with
450
IHV8ERT01EFD8V|lb 141 1.05 0.04 0.99 0.05 0.00 1.00 0.01 1.02 0.03 1.02 0.03 2.56 0.07 0.00 0.26 0.00 0.04…
but should start with
1779
IHV8ERT01EFD8V|lb 141 1.05 0.04 0.99 0.05 0.00 1.00 0.01 1.02 0.03 1.02 0.03 2.56 0.07 0.00 0.26 0.00 0.04…
I have modified mothur to correct this in the next release. In the meantime, changing the number of flows in your file will resolve the issue. Thanks for reporting this bug.
Kindly,
Sarah