Demux – QSeq demultiplexer

Demux is a Hadoop utility to demultiplex data from multiplexed Illumina runs.

Multiplexed runs are used to sequence multiple samples together. Each sample’s fragments are tagged with a barcode sequence, which is annotated in a sample sheet. Illumina’s base calling software then emits 3 reads for each DNA/RNA fragment:

1. read 1
2. barcode
3. read 2

Illumina provides a utility to separate the multiplexed reads by barcode, but it is a simple serial implementation. Seal Demux replaces this utility with a Hadoop-based implementation which can drastically reduce run times when run on a cluster and can leverage HDFS storage like the rest of the Seal tools.

Demux load the sample sheet and in particular keeps track of which lane/barcode combination is associated with each sample. For each flow-cell location present in the input, Demux gets the barcode (read 2), looks it up in the sample sheet and assigns the reads from that location to the appropriate sample.

Currently, Demux cannot handle errors in the barcode sequence. An error in the barcode will most likely result in the reads being placed in the “unknown” bucket.

Usage

To run Demux, you first need to copy the sample sheet to HDFS.

hadoop dfs -put sample_sheet.csv /user/me/

Then, run the bin/demux command in the Seal distribution:

./bin/demux --sample-sheet /user/me/sample_sheet.csv /user/me/qseq_input /user/me/demuxed_output

The arguments are:

1. --sample_sheet: the HDFS path to the sample sheet file;
2. Input paths: files or directories, containing files for reads 1, 2 and 3 in qseq format;
3. Output path: path to directory where demux will write its output. This directory must not already exist; demux will create it.

demux follows the normal Seal usage convention. See the section Program Usage for details.

Output

At the specified output path, demux creates a directory for each sample represented in the input. The directory name corresponds to the sample id in the sample sheet. Inside the sample’s directory you’ll find regular qseq files, where the records with read number 3 from the input have been shifted to read number 2.

In addition, you may find an unknown directory containing qseq files with all the reads whose barcode was not found in the sample sheet.

$ hadoop dfs -ls /user/me/demuxed_output
Found 5 items
drwxr-xr-x   - me supergroup          0 2011-06-23 17:17 /user/me/demuxed/_logs
drwxr-xr-x   - me supergroup          0 2011-06-23 17:29 /user/me/demuxed/snia_000186
drwxr-xr-x   - me supergroup          0 2011-06-23 17:29 /user/me/demuxed/snia_000268
drwxr-xr-x   - me supergroup          0 2011-06-23 17:29 /user/me/demuxed/snia_000269
drwxr-xr-x   - me supergroup          0 2011-06-23 17:29 /user/me/demuxed/unknown

Sample Sheet

The sample sheet is a table in comma-separated value format with the following colums, in order:

FCID:flow-cell ID Lane:flow-cell lane SampleID:name of the sample SampleRef:sample reference Index:barcode used to tag this sample, excluding the last base, A Description:whatever you want Control:N Recipe:experimental protocol Operator:operator name

Here’s an excerpt from a sample sheet file:

"FCID","Lane","SampleID","SampleRef","Index","Description","Control","Recipe","Operator"
"b02tgkkio",1,"csbb_001234","Human","ATCACG","Sequencing Project","N","tru-seq multiplex","Peter"
"b02tqacee",1,"csbb_004312","Human","CGATGT","Sequencing Project","N","tru-seq multiplex","Peter"

Counters

In addition to the counters from the Hadoop framework, Demux counts the number of reads found for each sample, and the unknowns. You’ll find them in the Sample reads counter group.

Configurable Properties

Demux does not have any program-specific configurable properties at the moment. You can still use its section to configure Hadoop property values specific to Demux.

Note

Config File Section Title: Demux