Axe Usage

Axe has several usage modes. The primary distinction is between the two alternate barcoding schemes, single and combinatorial barcoding. Single barcode matching is used when only the first read contains barcode sequences. Combinatorial barcoding is used when both reads in a read pair contain independent (typically different) barcode sequences.

For concise reference, the command-line usage of axe is reproduced below:

axe [-mzc2pt] -b (-f [-r] | -i) (-F [-R] | -I)
axe -h
axe -v

    -m, --mismatch	Maximum hamming distance mismatch. [int, default 1]
    -z, --ziplevel	Gzip compression level, or 0 for plain text [int, default 0]
    -c, --combinatorial	Use combinatorial barcode matching. [flag, default OFF]
    -p, --permissive	Don't error on barcode mismatch confict, matching only
                    	exactly for conficting barcodes. [flag, default OFF]
    -2, --trim-r2	Trim barcode from R2 read as well as R1. [flag, default OFF]
    -b, --barcodes	Barcode file. See --help for example. [file]
    -f, --fwd-in	Input forward read. [file]
    -F, --fwd-out	Output forward read prefix. [file]
    -r, --rev-in	Input reverse read. [file]
    -R, --rev-out	Output reverse read prefix. [file]
    -i, --ilfq-in	Input interleaved paired reads. [file]
    -I, --ilfq-out	Output interleaved paired reads prefix. [file]
    -t, --table-file	Output a summary table of demultiplexing statistics to file. [file]
    -h, --help		Print this usage plus additional help.
    -V, --version	Print version string.
    -v, --verbose	Be more verbose. Additive, -vv is more vebose than -v.
    -q, --quiet		Be very quiet.

Inputs and Outputs

Regardless of read mode, three input and output schemes are supported: single-end reads, paired reads (separate R1 and R2 files) and interleaved paired reads (one file, with R1 and R2 as consecutive reads). If single end reads are inputted, they must be output as single end reads. If either paired or interleaved paired reads are read, they can be output as either paired reads or interleaved paired reads. This applies to both successfully de-multiplexed reads and reads that could not be de-multiplexed.

The -z flag can be used to specify that outputs should be compressed using gzip compression. The -z flag takes an integer argument between 0 (the default) and 9, where 0 indicates plain text output (gzopen mode “wT”), and 1-9 indicate that the respective compression level should be used, where 1 is fastest and 9 is most compact.

The output flags should be prefixes that are used to generate the output file name based on the barcode’s (or barcode pair’s) ID. The names are generated as: prefix + _ + barcode ID + _ + read number + .extension. The output file for reads that could not be demultiplexed is prefix + _ + unknown + _ + read number + .extension. The read number is omitted unless the paired read file scheme is used, and is “il” for interleaved output. The extension is “fastq”; ”.gz” is appended to the extension if the -z flag is used.

The corresponding CLI flags are:
  • -f and -F: Single end or paired R1 file input and output respectively.
  • -r and -R: Paired R2 file input and output.
  • -i and -I: Interleaved paired input and output.

The barcode file

The barcode file is a tab-separated file with an optional header. It is mandatory, and is always supplied using the -b command line flag. The exact format is dependent on barcoding mode, and is described further in the sections below. If a header is present, the header line must start with either Barcode or barcode, or it will be interpreted as a barcode line, leading to a parsing error. Any line starting with ‘;’ or ‘#’ is ignored, allowing comments to be added in line with barcodes. Please ensure that the software used to produce the barcode uses ASCII encoding, and does not insert a Byte-order Mark (BoM) as many text editors can silently use Unicode-based encoding schemes. I recommend the use of LibreOffice Calc (part of a free and open source office suite) to generate barcode tables; Microsoft Excel can also be used.

Mismatch level selection

Independent of barcode mode, the -m flag is used to select the maximum allowable hamming distance between a read’s prefix and a barcode to be considered as a match. As “mutated” barcodes must be unique, a hamming distance of one is the default as typically barcodes are designed to differ by a hamming distance of at least two. Optionally, (using the -p flag), axe will allow selective mismatch levels, where, if clashes are observed, the barcode will only be matched exactly. This allows one to process datasets with barcodes that don’t have a sufficiently high distance between them.

Single barcode mode

Single barcode mode is the default mode of operation. Barcodes are matched against read one (hereafter the forward read), and the barcode is trimmed from only the forward read, unless the -2 command line flag is given, in which case a prefix the same length as the matched barcode is also trimmed from the second or reverse read. Note that sequence of this second read is not checked before trimming.

In single barcode mode, the barcode file has two columns: Barcode and ID.

Combinatorial barcode mode

Combinatorial barcode mode is activated by giving the -c flag on the command line. Forward read barcodes are matched against the forward read, and reverse read barcodes are matched against the reverse read. The optimal barcodes are selected independently, and the barcode pair is selected from these two barcodes. The respective barcodes are trimmed from both reads; the -2 command line flag has no effect in combinatorial barcode mode.

In combinatorial barcode mode, the barcode file has three columns: Barcode1, Barcode2 and ID. Individual barcodes can occur many times within the forward and reverse barcodes, but barcode pairs must be unique combinations.

The Demultipexing Statistics File

The -t option allows the output of per-sample read counts to a tab-separated file. The file will have a header describing its format, and includes a line for unbarcoded reads.