BarNone is usually used directly as a script, with the following options:
Two files are used for input: one containing the sequencing results, and another containing the barcode catalog file.
The sequencing file can be in any of the following formats:
The barcode catalog file should be tab-delimited, with content analogous to:
YBL103C AGTCTACCCACATGCTTTAG ATTCATAGGACACTTGCCGG
YBL104C ACTCATCAGGTATGGGAACG AATTCTAGCGCATGGTCCGG
YBL105C AGTGCTAATACGGCATAACG ACACTACCTAGATGGAGCGG
Each line has three components: a unique identifier for the gene, an uptag sequence, and a downtag sequence. The uptag and downtag sequences are each optional; if one is missing, it will be counted as having 0 reads in the output.
BarNone produces a tab-delimited file describing the number of reads that matched to each barcode.
If the file was not multiplexed, the output counts file has three columns, such as:
YAL008W 352 0
YBR255W 505 1411
YGR164W 429 1255
This describes, for each strain, how many up and down barcodes (respectively) were found.
If the file was multiplexed using the –multiplexfile option, the counts file contains two columns for each multiplexing barcode, and a header row describing each. For example:
Strain Sample1|UP Sample1|DOWN Sample2|UP Sample2|DOWN Sample3|UP Sample3|DOWN
YAL008W 352 0 471 0 478 0
YBR255W 505 1411 724 1319 754 1476
YGR164W 429 1255 608 1594 596 1495
BarNone is capable of constructing a new barcode catalog based on common mismatches found in a sequencing file. If a mismatched version of a barcode is found more frequently than the original, BarNone replaces that barcode with the more common version. This is specified using the –revisedcatalog option.
One can also obtain a much more detailed report about the specific mismatches using the –mismatchfile option. The specified file will be written in a format analogous to:
YOR041C ACATCCCGATCAGGTGACTG 23215 ACATCCCGATCAGGCGACTG (62)/CCATCCCGATCAGGTGACTG (34)/ACATCCCGATCAGNTGACTG (33)
YEL008W GCATCACAGCAATGGGCATA 2989 GCATCACAGCACTGGGCATA (20)/GCATCACAGCAACGGGCATA (8)/GCATCACAGCAATGGGCATC (8)
YMR011W GCCTCACTTAAAGCATACGA 6140 GCCTCACTTAACGCATACGA (33)/ACCTCACTTAAAGCATACGA (14)/GCATCACTTAAAGCATACGA (11)
YDR440W GCGGCCCTACAATTTATGAA 17 CCGGCCCTACAATTTATGAA (27840)/CCGGCCCTACACTTTATGAA (52)/ACGGCCCTACAATTTATGAA (46)
Each line contains the name of the strain, the original barcode, the number of times the original barcode appeared in the reads, and then a slash-delimited list of the close barcodes that were matched with the number of times each of them occured.
Of these four barcodes, YDR440W has a close match that is much more common than its original barcode, meaning that it would be replaced in a revised catalog.
Using the Gresham et al 2011 nutrient starvation data available here (in this example, the file Expt10.txt), would require a command like:
BarNone -f fastq -m 2 Expt10.txt counts.txt barcode_catalog.txt
This would match all barcodes in the file to a matching read in the catalog within an edit distance of 2 (set by the -m option).
An example of such a barcode catalog file is available in the examples/smith_et_al_2009_barcodes.txt.