In addition to demultiplexing, the Singular Genomics Demultiplex software can also filter the reads by FASTQ header flags as well as N-mask base calls where the PHRED quality score is below a predefined limit. This filtering/masking can occur during demultiplexing by including the following flags in the command line. Filtering and masking can also be applied to individual FASTQ files by using a dummy sample sheet.
Argument
Description
--quality-mask-threshold #
Template bases whose base quality is below the specified base quality threshold is output as a “N” rather than the original base call.
--filter-control-reads
Reads marked as control reads in their FASTQ headers are removed from the output FASTQ files. The G4 Sequencing Platform will perform real-time alignment to PhiX during the run, and mark reads aligning to PhiX as control reads in the FASTQ header
--filter-failing-quality
Reads marked as failing quality in their FASTQ headers are removed from the output FASTQ files. Currently, the G4 Sequencing Platform does not mark any reads as failing quality in the FASTQ header.
All 3 options are disabled by default in the standalone Singular Genomics Demultiplex software, meaning the software does not mask or filter out any reads by default. The flags must be included to enable the options. The onboard G4 Sequencing Platform demultiplexing does not filter any reads. However, the onboard G4 Sequencing Platform demultiplexing will N-mask bases with quality scores below 10 in reads longer than 35 bp. In reads less than 35 bp, no N-masking will occur. Please note that many downstream bioinformatic analysis software expects the N-masking threshold to be 2 rather than 10. You may need to adjust the N-mask threshold for better downstream compatibility.