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The trim.flows command is analogous to the trim.seqs command, except that it uses the flowgram data that comes bundled in the sff file that is generated by 454 sequencing. It's primary usage is as a preliminary step to running shhh.seqs. Chris Quince has a series of perl scripts that fulfill a similar role . This command will allow you to partition your flowgram data by sample based on the barcode, trim the flows to a specified length range, and cull sequences that are too short or have too many mismatches to barcodes and primers. For this page we use a hypothetical sff file - GQY1XT001.sff.
Preliminaries and defaults
To get going with trim.flows you first need to have flowgram data. You can generate this in mothur using mothur's version of sffinfo with the flows option.
mothur > sffinfo(sff=GQY1XT001.sff, flow=T)
Opening up GQY1XT001.flow in your favorite text editor will reveal a file that looks like this.
800 GQY1XT001CQL4K 85 1.04 0.00 1.00 0.02 0.03 1.02 0.05 ... GQY1XT001CQIRF 84 1.02 0.06 0.98 0.06 0.09 1.05 0.07 ... GQY1XT001CF5YW 88 1.02 0.02 1.01 0.04 0.06 1.02 0.03 ... GQY1XT001DB0ZG 85 1.04 0.00 1.01 0.01 0.02 1.01 0.00 ... GQY1XT001AKONJ 89 1.03 0.00 1.02 0.01 0.02 1.01 0.00 ... GQY1XT001BJEZH 292 1.03 0.01 1.00 0.02 0.03 1.02 0.00 ... GQY1XT001C1YUM 304 1.04 0.02 1.04 0.02 0.05 0.97 0.02 ... GQY1XT001BBRX0 140 1.02 0.03 0.99 0.03 0.04 1.05 0.04 ...
What you have on the first row is the total number of flow values - 800 for Titanium data. For GS FLX it would be 400. The second and following lines contain the sequence name, the number of useable flows as defined by 454's software, and the flow intensity for each base going in the order of TACG. So for example, the first sequence looks like....
GQY1XT001CQL4K 85 1.04 0.00 1.00 0.02 0.03 1.02 0.05 ...
By default, trim.flows will require each sequence to contain at least 450 flows and will trim each flowgram to 450 flows. We have found that this does the best job of reducing the sequencing noise. However, this assumes that your amplicon is shorter than 450 flows (i.e. Bacterial primers 27F/519R which amplify V1-3 only requires 350-400 flows to sequence depending on the exact sequence). Chris Quince suggests a minimum number of flows of 360 and a maximum of 720. We use Quince's settings for denoting the minimum signal (0.50) and noise (0.70). To run trim.flows with the default settings you will do the following:
mothur > trim.flows(flow=GQY1XT001.flow)
This will create a file called GQY1XT001.trim.flow and GQY1XT001.scrap.flow, that contain the trimmed files that passed the criteria and the sequences that didn't, respectively. In the GQY1XT001.scrap.flow file, you can see appended to the accession numbers the criteria(on) on which each read was culled (l for length, b for barcode, etc). Also created is a file called GQY1XT001.flow.files which lists all of the flow files that were created.
Rarely will you really just want the default settings. Below are some options that you are likely to want to use:
The oligos option takes a file that can contain the sequences of the forward and reverse primers and barcodes and their sample identifier. Each line of the oligos file can start with the key words "forward", "reverse", and "barcode" or it can start with a "#" to tell mothur to ignore that line of the oligos file. This same file can be used for running the trim.seqs command. For example, consider a trimmed version of sahl09.oligos:
forward CATGCTGCCTCCCGTAGGAGT vWhateverF #reverse TCAGAGTTTGATCCTGGCTCAG vWateverR barcode AACCAACC ALP50M barcode AACCAAGG AZAC1 barcode AACCATCG ALP2B barcode AACCATGC ALP1B barcode AACCGCAT ALP80M barcode AACCGCTA ALPG2 barcode AACCGGAA AZ273 ...
The forward primer is best thought of as the forward sequencing primer. So if you are using the 16S rRNA primers 27f and 338r to generate sequencing substrate, but you are sequencing off of the 338r end of the fragment, you would list 338r as the forward primer and 27f as the reverse. Here we are using a "#" for the reverse primer to indicate that we don't want mothur to screen for the reverse primer. If the "#" were removed, all of the sequences would wind up in the scrap file because the reads are typically not long enough to get to the distal primer. The lines starting with barcode follow the format of "barcode" - tab - barcode sequence - tab - sample identifier - line break. There is no limit to the number of primers or barcodes that mothur can handle. The forward and reverse primers can also be degenerate using standard IUPAC nomenclature. You can enter your oligos as upper or lowercase letters.
mothur > trim.flows(flow=GQY1XT001.flow, oligos=GQY1XT001.oligos)
Running trim.flows with the oligos file will generate a number of new flow files. For example:
GQY1XT001.A01.v35.flow GQY1XT001.A02.v35.flow GQY1XT001.A03.v35.flow GQY1XT001.A04.v35.flow GQY1XT001.A05.v35.flow GQY1XT001.A06.v35.flow GQY1XT001.A07.v35.flow ...
The GQY1XT001.flow.files file will contain a list of all of these files and will come in handy for the shhh.seqs command.
bdiffs & pdiffs & ldiffs & sdiffs & tdiffs
These parameters are used to allow differences in the barcode, primers, linkers and spacers. It has been shown that sequencing errors in the PCR primer region of a sequence correlate highly with poor sequence quality. Therefore, the default is to require an exact match to the primer or barcode sequences that you provide. pdiffs is maximum number of differences to the primer sequence, default=0. bdiffs is maximum number of differences to the barcode sequence, default=0. ldiffs is maximum number of differences to the linker sequence, default=0. sdiffs is maximum number of differences to the spacer sequence, default=0. tdiffs is maximum total number of differences to the barcode, primer, linker and spacer (default to pdiffs + bdiffs + ldiffs + sdiffs).
mothur > trim.flows(flow=GQY1XT001.flow, oligos=GQY1XT001.oligos, bdiffs=1, pdiffs=2)
minflows & maxflows
The minflows parameter will set the minimum number of flows that each sequence must contain to make it in to a "trim" file. By default this is set to 450; Chris Quince has preferred 360 in his documentation for processing GSFLX and Titanium data.
mothur > trim.flows(flow=GQY1XT001.flow, minflows=450)
The maxflows parameter will set the number of flows after which all other flows should be ignored. For instance, with Titanium data there are 800 flows. Setting maxflows to 450 (the default) will result in trimming the flowgram data to 450 flows. By default this is set to 450; Chris Quince has preferred 360 in his documentation for processing GSFLX data and 720 for Titanium data.
mothur > trim.flows(flow=GQY1XT001.flow, maxflows=450)
Combining minflows and maxflows allows you to set the range of flows you want. By default you will get flowgrams that are all 450 flows long. The following will give you Quince's recommended set up for Titanium data:
mothur > trim.flows(flow=GQY1XT001.flow, minflows=360, maxflows=720)
The fasta option allows you to tell trim.flows that you want it to translate the flowgram data to fasta sequence format:
mothur > trim.flows(flow=GQY1XT001.flow, fasta=T)
signal & noise
By default, trim.flows will treat any intensity signal greater than 0.50 as a real signal and any intensity less than 0.70 as noise. If an intensity falls between 0.50 and 0.70, it is treated as ambiguous and set as a trim point. The settings of 0.50 and 0.70 are suggested by Quince and we really see no need to change it, but in case people want to play with the values here is how you'd do it:
mothur > trim.flows(flow=GQY1XT001.flow, signal=0.60, noise=0.65)
Looking at the summary.seqs output for your dataset you may notice that the longest homopolymer in the dataset is 31 bases long. This is highly suspect as it is well-established that 454 technology struggles with homopolymers. To cap the homopolymer length you use the maxhomop option:
mothur > trim.flows(flow=GQY1XT001.flow, maxhomop=9)
It is likely that sequences with longer homopolymers will get culled for other reasons.
The order parameter is used to select the flow order. Options are A, B and I. Default=A, meaning flow order of TACG.
mothur > trim.flows(flow=GQY1XT001.flow, order=A)
The processors parameter allows you to run the command with multiple processors. By default processors is 1, and use of multiple processors is not available for windows users.
mothur > trim.flows(flow=GQY1XT001.flow, oligos=GQY1XT001.oligos, processors=2)
Putting it together
Here's how one might mix and match the settings to process their Titanium data...
mothur > trim.flows(flow=GQY1XT001.flow, oligos=GQY1XT001.oligos, pdiffs=2, bdiffs=1, processors=8)
This will generate individual flow files for each barcode/primer combination, GQY1XT001.trim.flow and GQY1XT001.scrap.flow files and a GQY1XT001.flow.files file.
- First Introduced - version 1.22.0.
- 1.23.0 - fixed bug that occurred if you had multiple primers with blank names. - http://www.mothur.org/forum/viewtopic.php?f=4&t=1358
- 1.24.0 - added linker and spacer option to the oligos file, as well as ldiffs and sdiffs parameters.
- 1.25.0 - allow for characters other than ATGC in reverse primers.
- 1.30.0 - added flow orders A, B and I.
- 1.33.0 - Bug Fix: printing trimmed number of flows to scrap file instead of original number of flows. Caused error if you wanted to read scrapped flow file.