How to concatenate the FASTQ files from different lanes

The following are options to concatenate the FASTQ files from different lanes of the same sequencing run on the NextSeq 500/550, HiSeq series, and NovaSeq 6000 instruments.

1. BaseSpace Sequence Hub

FASTQ Toolkit app and DRAGEN FASTQ Tookit app can be used to merge lane FASTQ files.

After selecting the input Biosamples and an Output Project, launch the app with default values for the settings in order to concatenate the lane FASTQ files. This results in the lane merged FASTQ files being aggregated within the original Biosamples. To prevent this automatic data aggregation, add a suffix with the 'Add a suffix to the output Biosample name(s)' setting; this outputs the merged FASTQ files to new Biosamples. More information about BaseSpace automatic data aggregation can be found here.

This app does require a small amount of iCredits due to the processing time.

2. bcl2fastq2/ BCL Convert conversion software

To automatically concatenate FASTQ files, add the following option to bcl2fastq command line.

--no-lane-splitting

This is described on page 15 of the bcl2fastq2 Conversion Software v2.20 Software Guide.

When BCL Convert is used for demultiplexing, adding the setting 'NoLaneSplitting, true' results in the creation of lane-merged FASTQ files. This is described in the BCL Convert online help page.

Note that the standalone version of BCL Convert works with both v1 and v2 sample sheets. while the BCL Convert app on BaseSpace only accepts v2 sample sheets as input.

3. Concatenate files after demultiplexing

3.1 Merge lane files using Window command prompt

Open Command Prompt, navigate to the folder containing FASTQ files (drag and drop the folder into the Command Prompt window) and type the following command.

copy /b *.fastq.gz Merged.fastq.gz

Note: /b is required to merge gzipped files, as it tells the copy program the files are binary and not plain-text.

3.2 Merge lane file using Linux

Use the following command.

cat file1.fastq file2.fastq > mergedfile.fastq

The standard cat command works fine, even in the gzipped state.

Note: Be aware that merged files, made with either method, cannot be uploaded to BaseSpace through the web file importer. To upload merged FASTQ files, use the BaseSpace CLI tool. For further information, search for Knowledge Base article How to upload FASTQ files to BaseSpace with BaseSpace CLI.

For any feedback or questions regarding this article (Illumina Knowledge Article #2035), contact Illumina Technical Support [e-mail].