DRAGEN FastQC

DRAGEN's FastQC module is a complete reimplementation of the original FastQC tool developed by the Babraham Institute (BI-FastQC). The reimplementation of FastQC in DRAGEN has been modified to take advantage of the hardware-acceleration provided by DRAGEN's Field-Programmable Gate Array (FPGA) for a significant speed improvement. There are some differences in how the values are calculated and the resulting metrics will not be identical between the two tools. The most significant differences are described below.

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Binning: BI-FastQC uses a customizable binning strategy with a default of 5bp bins, while DRAGEN uses an algorithmic binning strategy based on the Granularity setting resulting in DRAGEN providing more precise results at the default settings.

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Outputs: BI-FastQC text output contain the same information as their plots in tabular format, while DRAGEN-FastQC outputs it's raw data. For example, BI-FastQC both plots an outputs the average base quality per-position, while DRAGEN outputs the average base quality by both position and nucleotide. This allows for a more detailed analysis of the data, but requires slightly more work to generate the associated plot.

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Rounding: DRAGEN consistently rounds it's calculations to the nearest integer, while the original FastQC uses a mixture of rounding and taking the mathematical floor, leading DRAGEN-FastQC to provide incrementally higher results for some metrics.

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Smoothing: Both DRAGEN-FastQC and BI-FastQC utilize smoothing techniques for their distributions of %GC, to account for the fact that 151bp do not divide evenly into 100 percentile bins. To take advantage of the speed offered by the FPGA, DRAGEN utilizes a slightly different algorithm than BI-FastQC which results in slightly different results.

Due to memory constraints, it is not possible to guarantee single-base resolution for all metrics. DRAGEN provides an algorithmic solution for binning via --fastqc-granularity. DRAGEN allocates 256 bins in memory for each size or position-based metric. The granularity value of 4–7 inclusive can be used to determine the bin size. High values use smaller bins for greater resolution. Lower values can be used to create larger bins for larger read-lengths.

Granularity	Single Base Resolution (bp)	Resolution at 150 (bp)	Recommended Read-Lengths (bp)
7 (default)	1–255	1	< 256
6	1–128	2	≥ 256 and < 507
5	1–64	4	≥ 507 and < 4031
4	1–32	8	≥ 4031

To include metrics for adapter or other sequence content, DRAGEN FastQC needs the desired sequences to be provided in FASTA format. For this purpose, DRAGEN provides the following options for this purpose:

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For adapter sequences, use --fastqc-adapter-file.

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For any additional kmers of interest, use --fastqc-kmer-file.

With the --fastqc-kmer-file option, you can add sequences of interest without changing the expected adapter results.

DRAGEN FastQC can accept up to a combined total of 16 adapters and kmer sequences. Each sequence can be a maximum of 12 bp in length. By default, DRAGEN uses the adapter file located at /opt/edico/config/adapter_sequences.fasta. The file contains the following adapter sequences, which are the same as the FastQC from the Babraham Institute (v 0.11.10 and later).

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Illumina Universal Adapter—AGATCGGAAGAG

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Illumina Small RNA 3' Adapter—TGGAATTCTCGG

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Illumina Small RNA 5' Adapter—GATCGTCGGACT

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Nextera Transposase Sequence—CTGTCTCTTATA

The FastQC metrics are output to a CSV file format in the run output directory called <PREFIX>.fastqc_metrics.csv.

The reported metrics are organized into eight sections by metric type. Each section is categorized into separate rows by length, position, or other relevant categorical variables. The following metric types compose the sections.

Option	Description
Read Mean Quality	Total number of reads. Each average Phred-scale quality value is rounded to the nearest integer.
Positional Base Mean Quality	Average Phred-scale quality value of bases with a specific nucleotide and at a given location in the read. Locations are listed first and can be either specific positions or ranges. The nucleotide is listed second and can be A, C, G, or T. N or ambiguous bases are assumed to have the system default value, usually QV2.
Positional Base Content	Number of bases of each specific nucleotide at given locations in the read. Locations are given first and can be either specific positions or ranges. The nucleotide is listed second and can be A, C, G, T, N.
Read Lengths	Total number of reads with each observed length. Lengths can be either specific sizes or ranges, depending on the settings specified using --fastqc-granularity.
Read GC Content	Total number of reads with each GC content percentile between 0% and 100%.
Read GC Content Quality	Average Phred-scale read mean quality for reads with each GC content percentile between 0% and 100%.
Sequence Positions	Number of times an adapter or other kmer sequence is found, starting at a given position in the input reads. Sequences are listed first in the metric description in quotes. Locations are listed second and can be either specific positions or ranges.
Positional Quality	Phred-scale quality value for bases at a given location and a given quantile of the distribution. Locations are listed first and can be either specific positions or ranges. Quantiles are listed second and can be any whole integer 0–100.

The following examples include rows from each section.

Section	Mate	Metric	Value
READ MEAN QUALITY	Read1	Q38 Reads	965377
POSITIONAL BASE MEAN QUALITY	Read1	ReadPos 145-152 T Average Quality	34.49
POSITIONAL BASE MEAN QUALITY	Read1	ReadPos 150 T Average Quality	34.44
POSITIONAL BASE MEAN QUALITY	Read1	ReadPos 256+ T Average Quality	36.99
POSITIONAL BASE CONTENT	Read1	ReadPos 145-152 A Bases	113362306
POSITIONAL BASE CONTENT	Read1	ReadPos 150 A Bases	14300589
POSITIONAL BASE CONTENT	Read1	ReadPos 256+ A Bases	13249068
READ LENGTHS	Read1	150bp Length Reads	77304421
READ LENGTHS	Read1	144-151bp Length Reads	77304421
READ LENGTHS	Read1	>=255bp Length Reads	1000000
READ GC CONTENT	Read1	50% GC Reads	140878674373
READ GC CONTENT QUALITY	Read1	50% GC Reads Average Quality	36.20
SEQUENCE POSITIONS	Read1	'AGATCGGAAGAG' 137bp Starts	20
SEQUENCE POSITIONS	Read1	'AGATCGGAAGAG' 137-144bp Starts	23
POSITIONAL QUALITY	Read1	ReadPos 150 50% Quantile QV	37
POSITIONAL QUALITY	Read1	ReadPos 145-152 50% Quantile QV	37