TruPath Metrics

Reference guide for interpreting DRAGEN sequencing and proximity metrics and identifying potential performance issues.

Primary Metrics from RTA (Before DRAGEN Analysis)

The initial metrics obtained from the Real-Time Analysis (RTA) system prior to DRAGEN analysis are crucial for assessing sequencing performance. These metrics provide an indication of yield and sequencing quality across individual lanes and the entire flow cell.

Flow cell	%Q30	Yield/Lane	Yield/Total
C8	85%	241 Gb	1928 Gb
C2	85%	241 Gb	482 Gb

Contact customer technical support if any underperformance in yield or sequencing quality is observed whether across specific lanes or the entire flow cell. Reduction in yield and quality can be due to the DNA sample used, DNA input or user error; with other potential causes can be due to the reagent kits or instrument performance.

DRAGEN 4.5.2 Report

The performance ranges provided below are intended solely as guidance and do not constitute fixed specifications for these metrics.

For additional information on DRAGEN TruPath Reports, please visit the link here.

Summary Tab

• The metric ranges are tailored specifically to the TruPath genome workflow, whether using the C8 or C2 flow cell.

• Summary metrics are not influenced by DNA quality, but may be affected by sample type, extraction method, or workflow error.

• A lower percentage of unique reads correlates with decreased average coverage and proximity coverage in the resulting data. This does not correlate with a reduction in variant calling performance.

• Samples with unique read percentages below 70% are not classified as failed. Factors such as sample type, extraction method, and DNA input quantity may significantly impact unique read percentages and related metrics. Unexpectedly low results should trigger wider troubleshooting, including review of additional DRAGEN sample metrics, since multiple variables can affect these outcomes.

• Reduced unique reads are often associated with a larger median insert size.

• Lower DNA input, below minimum threshold of 175 ng, will likely result in lower unique read %, higher median insert size and lower average coverage.

Metric	Description	Expected Range	Notes
Average Coverage	Average alignment coverage across the genome	>50x	With one sample per lane, coverage may reach ~70x depending on % unique reads and % mapped reads. Lower coverage typically does not impact downstream variant calling, which is influenced by DNA quality and Q25 proximity coverage.
% >20x Coverage	Percentage of genome covered at ≥20x alignment depth	>93%	Indicates breadth of usable coverage across the genome.
%Q30 Bases	Percentage of bases with Q-score ≥30	>85%	Reflects overall sequencing base quality.
Input Reads	Total number of reads generated per sample	>1,600 M	Lower input reads may impact coverage and downstream metrics.
% Mapped Reads	Percentage of reads aligned to the reference genome	>94% (blood and cells only)	Saliva, buccal swabs, and other human sample types may contain non-human DNA, which can reduce mapped read percentages.
% Unique Reads	Percentage of reads remaining after duplicate removal	>70%	Lower values may indicate reduced DNA input or sample contamination. Samples below 70% are not classified as failed; unexpected values should prompt broader metric review.
% Proper Pairs	Percentage of reads mapped as properly paired	>85%	Reflects library quality and alignment consistency.
Median Insert Size	Median fragment insert size	18-400 bp	Driven by the on-flow-cell library preparation method; natural variation does not significantly affect proximity mapping or variant calling.

Proximity Tab

• The Proximity Tab in DRAGEN Reports is exclusive to the Illumina TruPath Genome, and provides insights on TruPath proximity related metrics.

• Proximity reads are those generated from the same original DNA fragment forming a template on the flow cell.

• All proximity metrics performance are closely linked to DNA quality and handling.

• Recommended DNA quality is 70% >10 kb and 40% >60 kb. Details on QC methods are available in the assay guide linked here.

• The following metrics indicate likely performance ranges if DNA quality and sample handling meet assay guide specifications. Lower metrics are expected with lower DNA qualities.

• If proximity metrics fall outside expected ranges but DNA quality is confirmed, DNA handling during sample addition and mixing may be the cause. Use wide bore tips and pipette slowly to minimize DNA shearing. Further information can be found in the assay guide linked here.

• The technical note linked here covers a wider range of sample input, lower quality DNA samples and associated metric ranges.

• Lower DNA input (below the minimum threshold of 175 ng) is likely to negatively impact Q25 proximity coverage. Q25 proximity rate and template length should remain equivalent or potentially increase slightly with significantly lower input DNA. Phasing Block NG50 can also be impacted negatively by significantly lower DNA input than recommended once the Q25 proximity coverage is below 30x.

Metric	Description	Expected Range	Notes
Fit RMSE	Proximity model fit error	<1	Reflects proximity model fit. Poor model fitting may occur in low-quality samples or samples with few proximity reads.
Q25 Proximity Rate	Percentage of proximity reads meeting Q25 quality threshold	40–75%	Values below range are most commonly associated with DNA below recommended quality. If DNA quality meets specifications, potential causes include DNA handling during sample addition and mixing; wide-bore tips and slow pipetting are recommended to minimize DNA shearing.
Q25 Proximity Coverage	Average coverage over genome with link-quality ≥Q25, on the phred scale	>30x	A reduction in average coverage and/or Q25 proximity rate can lead to a lower Q25 proximity coverage. These should be reviewed independently as described. Below 30x proximity coverage samples can most often still achieve expected variant calling performance; particularly for samples still meeting DNA quality expectations.
P75 Template Size	75th percentile proximity template length	15–100 kbp	Reduced template size is most strongly associated with sub-optimal DNA quality. If DNA quality meets specifications, DNA handling during sample addition and mixing may be contributory; wide-bore tips and slow pipetting are recommended.
Phase Block NG50	NG50 of phased haplotype blocks	700 kb – 35 Mbp	Lower values are most strongly associated with reduced DNA quality. Phase block NG50 varies substantially across HMW samples and may also be influenced by genome variation and regions of homozygosity.