Axe Usage


For arcane reasons, the name of the axe binary changed to axe-demux with version 0.3.0. Apologies for the inconvenience, this was required to make axe installable in Debian and its derivatives. Command-line usage did not change.

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

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

axe-demux [-mzc2pt] -b (-f [-r] | -i) (-F [-R] | -I)
axe-demux -h
axe-demux -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 prefix or existing directory]
    -r, --rev-in	Input reverse read. [file]
    -R, --rev-out	Output reverse read prefix. [file prefix or existing directory]
    -i, --ilfq-in	Input interleaved paired reads. [file]
    -I, --ilfq-out	Output interleaved paired reads prefix. [file prefix or existing directory]
    -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 index’s (or index pair’s) ID. The names are generated as: prefix + _ + index 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 index file

The index 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 indexing mode, and is described further in the sections below. If a header is present, the header line must start with either Barcode or index, or it will be interpreted as a index line, leading to a parsing error. Any line starting with ‘;’ or ‘#’ is ignored, allowing comments to be added in line with indexes. Please ensure that the software used to produce the index 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 index tables; Microsoft Excel can also be used.

Mismatch level selection

Independent of index mode, the -m flag is used to select the maximum allowable hamming distance between a read’s prefix and a index to be considered as a match. As “mutated” indexes must be unique, a hamming distance of one is the default as typically indexes 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 index will only be matched exactly. This allows one to process datasets with indexes that don’t have a sufficiently high distance between them.

Single index mode

Single index mode is the default mode of operation. Barcodes are matched against read one (hereafter the forward read), and the index 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 index is also trimmed from the second or reverse read. Note that sequence of this second read is not checked before trimming.

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

Combinatorial index mode

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

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

The Demultiplexing 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 reads which could not be demultiplexed.