HTG EdgeSeq Precision Immuno-Oncology Panel QC Metrics White Paper

For Research Use Only. Not for use in diagnostic procedures. HTG Molecular Diagnostics, Inc. In the U.S. and other applicable jurisdictions, HTG EdgeSeq is a trademark of HTG Molecular Diagnostics, Inc. 3430 E Global Loop, Tucson, AZ 85711 Any other trademarks or trade names used herein are the intellectual property of their respective owners. www.htgmolecular.com, 877-289-2615

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A Research Use Only QC Metrics White Paper for HTG EdgeSeq Precision Immuno-Oncology Panel

Introduction The HTG EdgeSeq Precision Immuno-Oncology Panel is a Research Use Only (RUO) gene expression profiling (GEP) assay that measures expression of mRNAs involved in oncology and immune pathways. The probe set for this assay comprises 1,410 nuclease protection probes, including 4 negative control probes (ANT), 4 positive control probes (POS), and 10 process control probes. Gene groupings include current drug targets, lymphocyte lineage markers, B, T, and NK cell activity markers, interleukin and chemokine markers, and proteasome-associated genes. Sample types for the assay include formalin-fixed, paraffin-embedded (FFPE) tissue or RNA derived from FFPE samples, as well as PAXgene samples.

This document describes a statistical method used to identify post-sequencing Quality Control (QC) failures within the HTG EdgeSeq Precision Immuno-Oncology Panel. These QC Metrics detect three sample failure modes: 1) degraded RNA or poor quality / quantity of sample; 2) insufficient read depth; 3) minimal expression variability across probes within a sample, wherein variation does not represent biological information. Three QC metrics (labeled as QC0, QC1, and QC2) were developed to target these three sample failure modes, respectively. Post-sequencing QC failures indicate sample, sample preparation, HTG EdgeSeq processor, or sequencing run issues and failed samples should be removed from subsequent analysis.

Study Design

A total of 288 samples were run in three plates for development of the QC0 and QC2 metrics. On each plate were FFPE samples from 4 cancer indications: Non-small cell lung carcinoma (NSCLC), melanoma, diffuse large B-cell lymphoma (DLBCL), and squamous cell carcinoma of the head and neck (SCCHN)), run in technical replicates (n=6). To mimic assay failure modes, a multi-tissue lysate (MTL) run with an eight-point titration of added EDTA (16, 8, 4, 2, 1, 0.5, 0.25 and 0 mM, six replicates each), and lysis buffer only (LBO) samples (n=24) were included in each of the three plates. Each plate was randomized separately using a distinct plate layout. For QC1 metric development, two samples - a mixed FFPE tissue control and a mixed cell line control - were run in triplicate.

Statistical Approach

1. QC0 development

The QC0 metric was developed to detect poor sample quality (degraded or low concentration). Poor quality samples tend to have little or no real biological signal. Each sample is evaluated by the percentage of read depth allocated to the 4 positive controls probes within the sample. The POS read depth is inversely proportional to the quality / quantity of the sample; i.e. samples with high sample quality result in low read depth for POS and vice versa. LBO samples serve as an experimental degraded / low-quality sample, as they are expected by their very nature to display little to no signal. Data from 72 LBO samples were utilized to establish a QC0 cut-off. Samples with a percentage of POS larger than the cut-off are deemed to be QC0 failures.

2. QC1 development

The QC1 metric was developed to detect samples with insufficient read depth. Down-sampling was used to determine the QC1 cut-off. Specifically, FASTQ files were “down-sampled,” or reduced such that they contained a random subset of fewer reads. The result of this down-sampling is a single sample that may be viewed at different read depths. Sequencing data from two samples (a mixed FFPE tissue control and a mixed cell line control) run in triplicate were used for this assessment. A range of 14 million to 10,000 reads was used. After the down-sampling was performed, the resulting FASTQ files were parsed. A log2 CPM of the parsed data was used to investigate the effect of the read depth on sample repeatability, and the QC1 cut-off was chosen at the read depth for which acceptable repeatability was observed for probes measuring low expressed (bottom of the range) genes. Sample repeatability was measured by intra-class correlation coefficient (ICC) through a mixed effect model. Sample repeatability was investigated using probes from 5%, 15%, and 30% quantiles of average expression.

HTG EdgeSeq Precision Immuno-Oncology Panel QC Metrics White Paper

For Research Use Only. Not for use in diagnostic procedures. HTG Molecular Diagnostics, Inc. In the U.S. and other applicable jurisdictions, HTG EdgeSeq is a trademark of HTG Molecular Diagnostics, Inc. 3430 E Global Loop, Tucson, AZ 85711 Any other trademarks or trade names used herein are the intellectual property of their respective owners. www.htgmolecular.com, 877-289-2615

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3. QC2 development

The QC2 metric detects samples with minimal expression variability across probes. Relative standard deviation (RSD) was used to assess the level of variation in expression throughout a sample. Less variation corresponds to a smaller RSD.

For each sample replicate (well), RSD is computed as:

𝑅𝑆𝐷 = %& ,

where 𝑠 and 𝑥 are the standard deviation and mean of log2(count+1), respectively.

EDTA inhibits the activity of S1 nuclease by chelating zinc (Zn2+) ions (Zn2+ is a required cofactor for the S1 nuclease used within the HTG EdgeSeq assay), which then artificially produces samples with minimal expression variability due to the presence of undigested probes.

Results

1. QC0 (percentage of POS)

Percentage of POS was computed for each sample / replicate across all 3 plates. As illustrated in Figure 1, there is a clear distinction between the LBO samples and FFPE (“true”) samples. As shown in Table 1, a cut-off of 40% provides a sensitivity of 95.8%, with only 3 of 72 LBO samples having less than 40% of counts allocated to POS. No true (non-LBO) sample had greater than 10% of counts allocated to POS.

The recommended QC0 cut-off is 40% of POS. Any sample replicate (well) resulting in greater than or equal to 40% of total reads allocated to POS probes is flagged as a QC0 failure.

Figure 1. The percentage of POS for all in the samples / wells from 3 plates. All LBO wells exhibited a high percentage of POS counts when compared to the percentage seen in “true” or FFPE samples.

Table 1. Sensitivity for different percentage of POS cut-offs

Cut-off Number of Failures Sensitivity

10% 0 100% 15% 1 98.6% 20% 2 97.2% 25% 2 97.2% 30% 2 97.2% 35% 3 95.8% 40% 3 95.8% 45% 4 94.4% 50% 4 94.4% 60% 5 93.1%

HTG EdgeSeq Precision Immuno-Oncology Panel QC Metrics White Paper

For Research Use Only. Not for use in diagnostic procedures. HTG Molecular Diagnostics, Inc. In the U.S. and other applicable jurisdictions, HTG EdgeSeq is a trademark of HTG Molecular Diagnostics, Inc. 3430 E Global Loop, Tucson, AZ 85711 Any other trademarks or trade names used herein are the intellectual property of their respective owners. www.htgmolecular.com, 877-289-2615

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2. QC1 (read depth)

Sample repeatability was investigated using probes from 5%, 15%, and 30% quantiles. Figure 2 demonstrates that as read depth increases, sample repeatability also increases. However, no additional improvements in repeatability were identified for read depths greater than about 2 million. It should be noted that data at a lower read depth is not necessarily unusable, but repeatability of the measurement of low expressing genes may be impacted.

Based on these results, a QC1 cut-off of 1.5 million counts is recommended. Any sample well with less than or equal to 1.5 million counts is flagged as a QC1 failure.

Figure 2. Intra-class correlation coefficient (ICC) for sample repeatability related to sample total read depth. As read depth increases beyond approximately 2 million reads, repeatability becomes stable.

3. QC2 (RSD)

The addition of EDTA at 4, 8, and 16 mM results in a bimodal RSD distribution, with lower EDTA addition and samples without EDTA having higher RSD values. As shown in Figures 3 and 4, the RSD from samples with EDTA added are smaller than the RSD from other samples. Based on the bimodal distribution of RSD, a QC2 cut-off at 0.15 is recommended. Any sample replicate (well) with an RSD value less than or equal to 0.15 is flagged as a QC2 failure.

Figure 3. Relative standard deviation (RSD) for each sample / replicate from all 3 plates. Samples with high additional EDTA added have a smaller RSD.

Figure 4. Distribution of RSD by plate. A bimodal distribution was observed in each plate. Samples / replicates with high additional ETDA have a smaller RSD.

Conclusion

Post-sequencing QC metrics (QC0, QC1, and QC2) for the HTG EdgeSeq Precision Immuno-Oncology Panel will detect three different sample failure modes as in Table 2. QC0 detects degraded RNA or poor quality / quantity samples. QC1 detects samples with insufficient read depth and QC2 detects samples with minimal expression variability. The developed QC metrics have high sensitivity for the identification of sample failures.

Table 2. HTG EdgeSeq Precision Immuno-Oncology Panel QC metrics

QC metric

QC failure mode Cut-off

QC0 Degraded RNA or poor quality

of sample

% of POS ≥ 40% as

failure

QC1 Insufficient read depth

Read depth ≤ 1.5M as

failure

QC2 Minimal

expression variability

RSD ≤ 0.15 as failure

HTG EdgeSeq Precision Immuno-Oncology Panel QC Metrics White Paper

For Research Use Only. Not for use in diagnostic procedures. HTG Molecular Diagnostics, Inc. In the U.S. and other applicable jurisdictions, HTG EdgeSeq is a trademark of HTG Molecular Diagnostics, Inc. 3430 E Global Loop, Tucson, AZ 85711 Any other trademarks or trade names used herein are the intellectual property of their respective owners. www.htgmolecular.com, 877-289-2615

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For more information, contact HTG Molecular Diagnostics, Inc. at 1-877-289-2615, info@htgmolecular.com or contact your local HTG representative. Visit www.htgmolecular.com