Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.


Introduction
The diagnosis and subclassification of cancers, fundamental to any cancer treatment algorithm, has historically been achieved through the study of the primary tumor tissue. The availability of high-quality diagnostic material suitable for high-end genomic technologies, including multiplexed tissue-based genotyping, is often limiting and associated with high risks and costs [1]. Indeed, for diseases such as advanced-stage, non-squamous, non-small-cell lung cancers (NSCLCs), approximately 30% of tumor samples yield insufficient or inadequate tissue for successful molecular genotyping [2,3]. Such limitations have positioned minimally invasive 'liquid biopsy'-based diagnostics as an attractive alternative for cancer management. Profiling of cell-free DNA (cfDNA) in blood to detect circulating tumor DNA (ctDNA) [4,5] has tremendous potential in overcoming the limits of sampling frequency and tumor accessibility, as well as the requirement of clinically overt disease imposed by traditional tumor biopsies.
Such advances are reflected in clinical practice upgrades, such as the National Comprehensive Cancer Network (NCCN) guidelines for NSCLC now encouraging the use of liquid biopsies in cases where tissue biopsies may not be feasible [6], particularly as many patients harbor methods [15,24] for single nucleotide variants (SNVs) and insertion/deletion events (INDELs), in the biologically relevant sub-1% ctDNA range. While multiple analytical and post-analytical factors differ between individual methods, including variant calling algorithms, better sensitivity can at least be partially attributed to the superior on-target capture rate of ampliconbased enrichment methods. At present, there is a paucity of comprehensive validation studies for NGS-based cfDNA tests, particularly those based on amplicon-based enrichment, which possess technical advantages in terms of simpler workflow, effectiveness with limited input DNA, and higher on-target capture rates.
In this work, we present the analytical validation of an amplicon-based NGS assay, LiquidHALLMARK®, with broad target coverage for clinically actionable genes. The assay incorporates two degrees of error-correction, through the use of unique molecular identifiers is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint

Assay design/specifications
LiquidHALLMARK is a targeted NGS-based sequencing diagnostic assay that profiles 80 cancer-related genes using cfDNA isolated from stabilized peripheral whole blood-derived plasma of cancer patients (Fig 1). The assay detects SNVs and INDELs in 77 genes, with nearly full exonic coverage in five genes and carefully curated inclusion of hotspot and critical regions in select oncogenes and tumor suppressors, spanning 58.8 kb of genome coverage. In addition, CNAs in 18 genes, and structural rearrangements in 10 genes, including the detection of both novel and pre-determined gene fusion events, are captured by the assay. Further, the assay detects MSI, as well as the presence of HBV and EBV, which are key oncogenic signatures in colorectal cancer, nasopharyngeal cancer, and liver cancer, respectively. Although less comprehensive than conventional 500-gene tissue-based NGS panels, rational inclusion of key cancer-specific and pan-cancer driver genes, non-genetic biomarkers, as well as targets for which FDA-approved drugs, investigational drugs, or clinical trials are available ( Supplementary Fig 1), has provided a diagnostic yield of 74.8% in all cancer samples processed to date (n = 1521).
LiquidHALLMARK utilizes a highly optimized amplicon-based library preparation method combined with UMI labelling to efficiently recover unique molecules from just 20 ng input cfDNA, sequencing to a median unique coverage of 8354X (range 42.0 -98.4% recovery of unique DNA molecules). With a median amplicon length of 166 bp, which approximates the median length of cfDNA [27], the assay generates highly uniform sequencing profiles; on average 97.1% of the panel has a depth no less than 0.2X the mean panel coverage and 79.0% of the panel has a depth no less than half the mean panel coverage. In addition, UMI sequencing combined with statistical background error-modelling approaches enable error suppression of more than 100X, allowing the confident calling of SNVs/INDELs above noise levels at as low as 0.01% variant allele frequency (VAF; Supplementary Fig 2).
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SNV/INDEL detection performance
To determine the analytical sensitivity, precision, and accuracy of the LiquidHALLMARK assay in detecting SNVs and INDELs, the reference standard sets HD780 (Fig 2A) and diluted Tru-Q (Fig 2B; Horizon Discovery), comprising a total of 39 unique clinically relevant SNVs and three INDELs in 19 genes across 5% (3.6 -6.5%), 1% (1.0 -1.3%), and 0.1% (0.10 -0.13%) VAF verified by droplet digital PCR (ddPCR), were run in triplicates across multiple operators, two reagent lots, and two NextSeq 550 sequencers. In addition, the HD786 reference standard  Table 1). The per-base analytical specificity prior to noise filtering was determined to be 99.98% at 5% and 1% VAF, and 99.11% at 0.1% VAF using a total interrogable panel size of 58.8 kb, giving a false positive rate of 0.022% at 5% VAF, 0.025% at 1% VAF, and 0.90% at 0.1% VAF. These results confirm a 90% limit of detection (LoD90) of 0.1% VAF for both SNVs and INDELs. Across the HD780 reference standards, pairwise analysis gave an overall reproducibility of 100% at 5% and 1% VAF, and 97.22% at 0.1% VAF, with no noticeable difference within or between operators, reagent lots, and sequencers (Fig   2A). Quantitative accuracy of variant calls was high, with a good degree of correlation between observed and expected VAFs (Fig 2C; R  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  (Fig 2D-E), with good agreement between quantitative calls made by both methods (Fig 2F). In total, four exon 19 deletion, one L858R, and one T790M variants were missed by LiquidHALLMARK at detected VAFs close to or below the LoD90 (AS-PCR VAF 0.03 -0.16%). LiquidHALLMARK detected three additional G719X, four exon 19 deletion, five L858R, and five T790M variants (NGS VAF 0.02 -0.50%); 10 of these were detected specifically by AS-PCR melt curve analysis below the reportable range. Of the remaining seven discordant variants, six were detected close to or below the established LoD of AS-PCR, with one discordant exon 19 deletion (L747_P753delinsS) detected at 0.5% VAF in LiquidHALLMARK.
These findings corroborate the high sensitivity and accuracy of the LiquidHALLMARK assay.
Due to a lack of fusion-positive genetic material, the platform technology was further validated for the ability to detect novel fusions by analyzing 29 NK/T-lymphoma tissue samples for PD-L1 3'-untranslated region (3'-UTR) rearrangement positivity [29]. Of these, nine samples tested positive for PD-L1 3'-UTR rearrangement and 20 samples tested negative; all calls were orthogonally confirmed by whole-genome, targeted, or Sanger sequencing, giving an overall concordance of 100% (Supplementary Table 1). In one sample where matched plasma was available, cfDNA testing identified the same PD-L1 3'-UTR rearrangement. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint

Copy number alteration (CNA) detection performance
Validation of CNA detection performance was conducted using contrived admixtures of fragmented cell-line DNA from cell-lines well-established to harbor gene amplifications in EGFR, ERBB2 (HER2), NRAS, and CDK6 [30-32], as well as the HD786 reference standard which harbors focal amplification in MET and MYC at 4.5 and 9.5 copies respectively, instead of normal diploid two copies. Across 19 observations from 2.4 to 65.2 gene copies in the dilution series, all amplifications were positively detected, with a high degree of correlation between expected and observed copy numbers (R 2 = 0.92, y = 0.70; Fig 3A). Based on gene amplifications confidently detectable above the range of normalized copy numbers determined from a set of selected reference plasma cfDNA samples (n = 27), the LoD for gene amplifications was determined to be a 1.5X increase from the normal diploid number, which is equivalent to a low-level amplification (copy number of 6) at 25% tumor fraction (TF) or a high-level amplification (copy number of 10) at 12.5% TF.
To assess the concordance between gene amplification calls in plasma cfDNA and fluorescence  Table 2).

Detection performance for other biomarkers
In addition to detecting conventional biomarkers in SNVs, INDELs, gene fusions, and CNAs, the LiquidHALLMARK assay detects and quantifies viral loads of EBV and HBV in plasma cfDNA via the inclusion of amplicons specific for these viruses. EBV detection performance was verified by spiking plasma cfDNA with known viral loads of the external reference standard for EBV (1 st WHO International Standard for Epstein-Barr Virus, NIBSC code: 09/260) spanning six orders of magnitude. Based on the dilution series performed in four replicates, a high degree of linearity could be observed for all eight EBV-specific amplicons (R 2 >0.95 for all amplicons, Fig 3B). These experiments also establish an LoD of 10 IU EBV within a sequencing run, equivalent to 2 IU/mL plasma when the routine 5 mL of plasma is used for cfDNA is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  Table 3).
HBV detection performance was assessed by spiking plasma cfDNA with known viral loads of the external reference standard for HBV (4th WHO International Standard for HBV DNA for NAT, NIBSC code: 10/266) spanning six orders of magnitude. Based on the dilution series performed in four replicates, a high degree of linearity of response was observed across both HBV-specific amplicons (R 2 >0.98 for both amplicons, Fig 3C).
The detection threshold for microsatellite instability (MSI) was determined using 91 plasma cfDNA samples and 29 FFPE tissue samples with no known MSI, with the threshold for calling MSI set as the 95 th percentile of deletion length (for each of the six MSI loci) detected across all microsatellite stable (MSI-negative) samples. The LoD for MSI was validated using dilutions of DNA from the MSI-positive cell-line RKO in a wild-type cfDNA background and confirmed to be 5% tumor fraction (Supplementary Fig 3). To  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  Fig 4A), with 1 EGFR L858R detected by cobas but not LiquidHALLMARK.
To resolve the true nucleotide identity of this potential false negative variant, plasma cfDNA from this sample was orthogonally tested by AS-PCR, which corroborated the absence of EGFR

Clinical utility of the LiquidHALLMARK Assay
While validation studies are crucial for examining the technical performance of an assay, insight into the performance of an assay in a real-world setting is equally, if not more, informative on the utility of a liquid biopsy test in guiding clinical decisions. To that end, we examined all samples submitted to our CAP-and CLIA-accredited laboratory to elucidate realworld assay performance metrics. Within the study period from January 2018 to May 2021, a total of 1,592 samples were analyzed and reported (100% report rate), with 52.1% of the clinical volume originating from lung cancer patients (Fig 5A). While the assay is intended for . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint advanced cancer patients to aid clinical therapeutic decision making, a significant portion (n = 71; 4.5%) of the clinical volume comprised screening cases in healthy individuals, or suspected cancer cases. Additionally, samples from patients with localized tumors constituted 8.7% of the clinical volume. Overall, 73.6% (1120/1521) of cancer samples harbored at least one detectable genetic variant (ctDNA positive), including 40.6% of samples from localized tumors and 78.5% of samples from metastatic tumors. When non-genetic biomarkers (EBV/HBV/MSI) were included, the detection rate increased to 74.8% (n = 1137). While ctDNA detection was largely uniform across most tumor types, several cancers such as kidney cancer and melanoma displayed lower ctDNA detection rates (Fig 5A), consistent with previous reports on tumor type-specific ctDNA prevalence [15]. Among ctDNA-positive samples, a median of two variants were detected per sample, with a median VAF of 0.86% (range 0.01 -95.30%).
Additionally, more than half of ctDNA-positive cancer samples contained at least one variant ≤0.3% VAF; this was again consistent across tumor type, highlighting the ability of the assay to sensitively detect variants at sub-1% VAFs. Across all non-cancer screening samples (n = 61), two likely pathogenic SNV variants (JAK2 V617F, detected at 0.81% VAF, and TP53 M246I, is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint having a TF ≤0.3% (19.4% in lung cancer samples; Fig 5C), emphasizing the importance of high sensitivity in liquid biopsy assays.

Clinical actionability
A cornerstone of NGS-based liquid biopsy assays is the ability to detect clinically actionable alterations not easily detected via alternative testing methods. Hence, we further analyzed the prevalence of driver genes and clinically relevant variants detected within the 1,592 clinical samples. In line with known prevalence of significantly mutated genes in cancer [41,42], TP53, KRAS, PIK3CA, APC, SMAD4, and PTEN were among the most frequently altered genes across all ctDNA-positive samples (Supplementary Fig 4A). In addition, EGFR alterations were detected in 36.1% of all ctDNA-positive samples; this was driven by the large proportion of lung cancers among clinical samples. Indeed, in ctDNA-positive lung cancer samples, the detection rate of EGFR alterations was 62.8%, with more than a third of detected EGFR alterations being in-frame exon 19 deletions (Fig 5D, Supplementary Fig 4B). Significant alteration frequencies were also observed in a repertoire of clinically relevant genes including KRAS, PIK3CA, MET, ERBB2, BRAF, and ALK, as well as driver genes with potential actionability such as NF1, KEAP1, and NFE2L2 (Fig 5D). Importantly, of the 615 ctDNApositive lung cancer samples, 83.1% (n = 511) harbored at least one genetic biomarker possessing biological evidence for actionability, including 72.5% (n = 446) with ≥1 biomarker with a guideline-approved drug, and an additional 4.2% (n = 26) and 6.3% (n = 39) harboring either biomarkers predicting drug resistance or with biological evidence suggesting potential clinical actionability (both drug sensitivity and resistance) respectively (Fig 5E top).
Sensitizing EGFR mutations (EGFR L858R, exon 19 deletion, or L861Q/G719X/S786I) were by far the most prevalent druggable target, being observed in 78.5% of lung cancer samples with guideline-approved biomarkers, while EGFR T790M, exon 20 insertions, and amplifications, were also observed at significant frequencies (Fig 5E bottom). Beyond drug sensitivity, the prevalence of alterations that predict treatment resistance was also further analyzed. On-target is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  (Fig 5F). Strikingly, 32.8% (22/67) of all EGFR T790M resistance mutations in firstor second-generation EGFR TKI-treated patients were detected at VAF ≤0.3%, while 42.6% (26/61) of all resistance mutations detected in osimertinib-treated patients were observed at VAF ≤0.3%. In breast cancer, clinically relevant alterations in PIK3CA, ESR1, BRCA2, and ERBB2 were among the most prevalent (Fig 5G, Supplementary Fig 4C) Figure 5B). Across the three cancer types, actionable variants were among the most frequently observed variants, with VAFs ranging from 0.01 -94.30% (Table   4). Collectively, these results demonstrate the capability of LiquidHALLMARK to guide clinical therapeutic decisions.

Molecular profiling to delineate origins of cancers of unknown primary
Broad genomic coverage also aids the molecular profiling and differential diagnosis of cancers of unknown primary (CUP). Of the 36 CUP cases analyzed, 80 unique genetic alterations across 34 genes were detected in 30 cases, giving a ctDNA detection rate of 83.3% ( Supplementary   Fig 6). Detection of tumor type-specific alterations, including AR, ESR1, GNAS, and EGFR alterations in seven samples, as well as EBV, HBV, and MSI positivity in three samples, informed potential tumor origins. Within genes with high prevalence of mutations, tumor specific prevalence biases based on the COSMIC database [51] could be used to discriminate between tumor types. For instance, several TP53 alterations identified favored a large intestinal origin (R273C, R273H), while others favored a lung origin (K305*, V157F, R110P). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint

Cancer monitoring through alternative non-genomic biomarkers
In tumor types where the prevalence of conventional genetic biomarkers is low, such as nasopharyngeal cancers (NPC), viral load quantification acts as a useful marker of tumor burden [52]. Of the NPC samples analyzed, 64.3% (9/14) were ctDNA positive, with TP53 alterations being the most prevalent. By contrast, 69.2% (9/13; one sample was not tested for EBV) of NPC samples were EBV positive (Supplementary Fig 7A), including two ctDNAnegative samples, enabling an additional diagnostic yield of 14.3%. In liver cancer, although 85.0% (17/20) of samples were ctDNA positive and only 55.6% (10/18; 2 samples were not tested for HBV) of samples were positive for HBV (Supplementary Fig 7B), one ctDNAnegative sample was HBV positive, providing an additional diagnostic yield of 5.0%. The detection of microsatellite instability also contributed to diagnostic yield; 16 samples were identified as MSI positive, including six MSI-high samples and 10 MSI-low samples. Of these, three were negative for any ctDNA-based genetic variant.

Discussion
Tissue biopsy has historically been the gold standard of cancer diagnostics, enabling the histopathological analysis of tumors, and since the advent of precision oncology, facilitates the molecular profiling of tumors to inform therapeutic decisions [53]. Yet, as the field of molecular biology advances, the limitation of tissue biopsy for molecular profiling becomes increasingly evident. Critically, the isolated snapshot provided by a single tissue biopsy is unable to wholly recapitulate a tumor, particularly given extensive evidence of intratumoral heterogeneity, which may impact therapeutic options [54][55][56]. This emphasizes the need for serial biopsies, an option that is often infeasible given the invasive nature of tissue biopsies and their accompanying clinical risks, potential surgical complications, and economic costs [57].
In scenarios where tumor accessibility is an issue, obtaining even a single tissue biopsy can be onerous [53]. In light of this, liquid biopsy presents a minimally invasive opportunity to interrogate tumor biology without additional risks to patients, enabling early cancer detection, prognostication, and disease monitoring through a simple blood draw. Several cfDNA-based liquid biopsy companion diagnostics currently exist; these assays probe specific molecular targets to predict drug sensitivity, for instance, EGFR in the cobas EGFR Mutation Test v2 and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint 5.7% and 8.1% harboring biomarkers associated with treatment resistance and potential actionability respectively. Importantly, the diverse spectrum of alterations across the 80 cancerrelated genes covered in the panel is highlighted by the 1607 unique somatic variants identified across all clinical samples, emphasizing the necessity of broad genomic coverage.
Underpinning the high sensitivity of the assay is the frequent detection of samples with TFs ≤ 0.3%, with variants routinely detected below the LoD; indeed, 10.2% of all reported variants were found at VAFs between 0.01% and 0.09%, with calls being supported by high variant allele read counts and a high depth of coverage. In lung cancer, the assay identified an actionable biomarker in 83.1% of ctDNA-positive cases, including 28.5% of cases where the actionable biomarker was detected at a low VAF of ≤0.3%. Significantly, nearly a third of first-or secondgeneration EGFR TKI-associated EGFR T790M resistance mutations, and more than two-fifths of osimertinib-associated resistance mutations, were detected at low VAFs ≤0.3%. These were detected at 0.81% and 0.40% VAF respectively, demonstrating a per-sample specificity of 96.72% or per-base specificity of ≥99.9999%. Furthermore, given that JAK2 V617F and mutations in TP53 are two of the most common somatic alterations detected in plasma cfDNA due to clonal hematopoiesis (CH) [29], the possibility that these two mutations arose due to CH cannot be ruled out. Taken together, these results underline the excellent sensitivity and specificity of the LiquidHALLMARK assay within a real-world clinical setting. In addition, the inclusion of less commonly-targeted oncogenic biomarkers in LiquidHALLMARK enhances diagnostic yield. For instance, EBV clearance in ctDNA has been investigated as a surrogate for clinical outcomes in the only prospective, randomized, interventional study in the minimal residual disease (MRD) setting [58] and an EBV qPCR assay has also been used as a bloodbased primary screening test for nasopharyngeal cancer [59], while MSI is an actionable biomarker that is routinely tested in tumor tissue but is currently limited in liquid biopsy assays. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint EBV and HBV detection enabled a further diagnostic yield of 14.3% and 5.0% respectively in nasopharyngeal and liver cancers, while MSI detection provided on-label drug recommendations in six samples and additional diagnostic yield in three ctDNA-negative samples.
Collectively, these results establish the performance of an amplicon-based liquid biopsy assay in a niche where the utility of hybrid-capture methods has been much more extensively evaluated [15,24]. Although conventional whole-exome amplicon-based NGS sequencing has been associated with lower uniformity of depth [60], this limitation is easily circumvented in targeted liquid biopsy panels by rational design, particularly given that cfDNA is fragmented in a non-random fashion [61][62][63]. Indeed, LiquidHALLMARK demonstrates highly uniform  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint

Sample collection and processing
Two 10 mL tubes of whole blood were collected for each patient in Streck Cell-Free DNA BCT (Streck), shipped to Lucence Diagnostics at ambient temperature, and processed within 6 -48 h of sample collection. Upon sample receipt, samples were accessioned and both 10 mL tubes of whole blood per patient were fractionated to isolate plasma via centrifugation at 1,600 g for 10 min at 4 o C. Isolated plasma was clarified via further centrifugation at 16,000 g for 10 min at 4 o C before being aliquoted and stored at -20 o C or processed immediately for cfDNA extraction.

Bioinformatics processing and variant calling
Illumina binary base call sequencing files were demultiplexed and converted to fastq file format using the bcl2fastq v2.20 software (Illumina) before being processed with an in-house is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint bioinformatics pipeline (Lucence Diagnostics) for base quality filtering (Phred quality score <15), adapter trimming, and consensus UMI clustering. Consensus reads were then aligned to the hg19 reference genome using BWA-MEM v0.7.17 [64]. For SNV and INDEL detection, consensus reads were compared against position-and sequence run-specific noise level profiles trained using a curated selection of 110 past samples run through the in-house pipeline to estimate the probability of noise for each variant in a sample. Position-specific pvalue cutoffs were used to discriminate between true and noise variants. All true variants found within coding sequences and splice sites, as well as pathogenic variants within promoter regions based on ClinVar classification were reported [65]. For fusion detection, paired-end reads were mapped to the reference genome and alignment information including directionality and soft-clipped sequences were analyzed to identify potential fusion events. To detect CNAs, probe-level consensus coverage was self-normalized to a set of invariant genomic positions and compared against the normalized coverage distribution constructed from baseline samples before being aggregated to the gene level (z-score) [66]. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

Limit of detection
The lower limit of detection is defined as the lowest VAF at which an alteration can be consistently measured ≥90% of the time (LoD90), and was determined empirically based on all assay replicates run on all reference standard materials (HD780, HD786, Tru-Q, and cell-line is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. CNAs were analyzed in 12 -18 genes, and structural rearrangements were analyzed in 3 -10 genes, depending on the assay version used (see Supplementary Fig 1).

Clinical and orthogonal validation
The reference AS-PCR method [28] used for orthogonal EGFR variant detection was performed in-house using variant-specific PCR primer pairs with wild-type-or mutantspecific fluorescent probes for 10 pre-determined EGFR variants. The limit of detection (LoD) for the reference AS-PCR method is 0.05% VAF for EGFR exon 19 deletions (E746_A750del, L747_P753delinsS, L747_A750delinsP, and L747_T751del) and L861Q, 0.1% VAF for EGFR G719A, G719S, T790M, and L858R, and 0.5% for EGFR G719C. For external validation of EGFR variants, 2 mL of plasma cfDNA from deidentified clinical samples were submitted to the Mayo Clinic Laboratories (Rochester, MN) for cobas EGFR Mutation Test v2. Orthogonal EBV detection was performed by PCR as previously described [33].
Contingency tables were constructed to calculate positive percent agreement (PPA), negative percent agreement (NPA), and overall concordance using standard methods [67]; 95% confidence intervals (95% CI) were calculated using a hybrid Wilson/Brown method [68]. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ;   is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ;

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. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted August 8, 2021. ; https://doi.org/10.1101/2021.08.03.21261575 doi: medRxiv preprint