NS of 160 bp vs 10 kb fragmented human genomic DNA. Fig. S1 shows gel images of human genomic DNA fragmented to mean length of 160 bp or 10 kb. NS read statistics of the two libraries are shown in Fig. S and Fig. S . The mean read length of 160 bp fragmented library was 265 bp, due to the attachment of barcodes and sequencing adapter prior to NS. The 10kb DNA library had a mean read length of 3.8 kb. The Q-score of 10 kb library was higher than that of the 265 bp library. 

Section S2: SAL Design and Performance Concatamer (0% VAF) Amplicon (0% VAF) FIG. S . NS amplicon t races with and without SAL. T op panels s how amplicon t races f or 0% V AF s ample. Bottom panels s how amplicon traces for 5% VAF sample. VRF of NS reads at each location that corresponds to the highest frequency single-base changes at that position. VRF in Fig. 2e was calculated by subtracting VRF of 0% VAF sample from 5% VAF sample. 

Section S3: Bioinformatic Analysis

Summary of bioinformatic workflow. NS on MinION was run for 30 min to 1 hour depending on the number of barcoded samples. Typically, 350k-500k reads were obtained in the first one hour of NS. Base-called reads were demultiplexed using EPI2ME software (Oxford Nanopore Technologies). Depending on the total number of reads obtained per sample, reads were subsampled randomly to approximately 10,000 reads per sample for analysis. The NS reads were then deconcatenated using a custom python script. 
The python script used minimap2 (github.com/lh3/minimap2) to map individual wildtype amplicon reference sequences to the concatenated reads and extract the mapped monomer sequences from the concatenated read. This step also removed any off-target amplicon sequences from the concatenated reads. Fig. S shows a sample concatenated read. 
The mapping efficiency for finding monomer sequences within concatenated reads was calculated as mapped nucleotide fraction (MNF), the fraction of concatemer reads that mapped to an amplicon of interest (Fig. S ). Manual analysis of concatenated reads showed that minimap2 missed some monomers (Fig. S ). Tables S1 andS2 shows the number of reads analyzed for each sample and the MNF values. MNF was higher for FFPE samples, which could be due to the fragmented nature of DNA that resulted in reduced amplification of off-target sequences. Sub-sampling different sets of approximately 10,000 reads resulted in same variant calls with similar % VRF values (Table S3). 
The mapped on-target sequences obtained after deconcatenation were then analyzed in two ways for variant calling. First, the sequences were aligned to amplicon reference sequences using minimap2 aligner to generate a BAM alignment file. IGVtools was used on the BAM file to extract the number of A, C, G, and basecalled nucleotides, insertions and deletions at each position using the basecount command. The VRF at each nucleotide position was calculated as the highest frequency single-base change at that position. Positions with VRF ¿20% were flagged as potential variants. 
Second, the sequences were aligned to human reference genome (GRCh38) using minimap2 aligner to generate a BAM alignment file. The BAM file was down-sampled to <150x coverage for each amplicon using samtools view command. The downsampled BAM file was then used with Clair to call variants. Variant calls with score > 180 were flagged as potential variants. The bioinformatic workflow for variant calling using Clair variant caller is summarized in Fig. Section S4: Analytical Validation Experiments with Synthetic DNA 
Analytical validation experiments using synthetic DNA spike-in samples. Multi-gene OCEANS panels were calibrated by a spiking synthetic mutation-bearing DNA oligonucleotide (gBlock) into the human NA18562 gDNA. The 10% VAF positive control sample had its VAF confirmed by NGS, and was diluted with NA18562 gDNA to prepare positive control samples with VAF ranging between 1% and 0.05%. For 1% and 0.5% VAF samples, 50 ng input DNA was used for OCEANS panels, corresponding to 15,000 haploid copies. For 0.2%, 0.1%, and 0.05% VAF samples, 100 ng input DNA was used for OCEANS panels. 
OCEANS reads were deconcatenated, aligned to human reference genome (GRCh38), and then both the Clair score and the Variant Read Fraction (VRF) were calculated for each mutation. A summary of the variant calls made by Clair for melanoma OCEANS panel's calibration runs is shown in Enrichment fold (EF) calculation. Enrichment fold was calculated as described previously in ref. 
[1]. Briefly, EF was calculated from the variant read fraction (VRF) observed from NS after OCEANS and the variant allele fraction (VAF) in the original sample based on the formula: 
EF = ( 1 V AF V AF ) ( 1 V RF V RF )
For the AML and melanoma panels, EF was calculated for each plex by spiking synthetic DNA bearing a mutation into NA18562 gDNA shown in Fig. 4a and Fig. 4d. Median EF was calculated from EF values in the linear range from 0.05% to 1% VAF for each plex. Given a EF value for a particular mutation, the original VAF of the mutation in the sample can, in principle, be calculated from the VRF based on the formula 
V AF = V RF . (EF 1)(1 V RF )+1
However, due to the relatively high variation in observed VRF due to nanopore sequencing's intrinsic error rate, the effective dynamic range of quantitation is small, and VAF estimations are accurate only when the observed VRF is between 10% and 90%. For example, given an EF value of 1000, 99% VRF and 95% VRF correspond to VAFs of 10% and 2%. At 85% vs. 80% VRF, the VAFs correspond to 0.56% and 0.40%, which is a relatively smaller difference. 
Mutation detection without SAL. AML OCEANS panel was run on 1% VAF synthetic DNA spike-in samples without SAL. Enrichment of variants were similar to OCEANS panel with SAL (Fig. S4-2). However, the throughput without SAL was around 6-fold lower. Therefore, BDA alone will be able to detect mutations as low as 1% VAF while SAL significantly improves throughput for mutation detection. 

Gene

Enrichment Region (GRCh38) FLT3 
Chr13 : 28,018,018,513 DNMT3A Chr2: 25,234,234,383 IDH1 Chr2: 208,248,248,413 KIT Chr4: 54,733,733,168 NPM1 Chr5: 171,410,410 : 66,435,435,129 MAP2K1 Chr15: 66,436,436,830 MAP2K1 Chr15: 66,481,481,804 MAP2K2 Chr19: 4,117,117,558 MAP2K2 Chr19: 4,110,110,588 MAP2K2 Chr19: 4,101,101,106 AKT1 Chr14: 104,776,776,714 AKT3 Chr1: 243,695,695,726 NRAS Chr1: 114,716,716,137 NRAS Chr1: 114,713,713,912 KRAS Chr12: 25,245,245,358 KRAS Chr12: 25,227,227,346 PIK3CA Chr3: 179,218,218,309 PIK3CA Chr3: 179,234,234,307 BRAF Chr7: 140,753,753,353 : 55,191,191,831 KRAS Chr12: 25,227,227,356 KRAS Chr12: 25,245,245,352 KRAS Chr12: 25,225,225,628 MAP2K1 Chr15: 66,435,435,124 MET Chr7: 116,771,771,998 NRAS Chr1: 114,716,[111][112][113][114]716,127 NRAS Chr1: 114,713,713,924 PIK3CA Chr3: 179,218,218,311 PIK3CA Chr3: 179,234,234,301 PTEN Chr10: 87,957,957,922 ROS1 Chr6: 117,318,318 shows the location of TERT promoter mutation in a homopolymer region. FF and FFPE samples where insufficient amounts of DNA (< 5ng) were extracted were excluded from these lists, and OCEANS panels were not run on them. Table S shows clinical sample information for melanoma FF samples; FFPE sample clinical information were not available. Table S andTable S show clinical sample information for NSCLC and HCC FF and FFPE samples. 
There are significantly higher number of mutations calls in FFPE samples compared to FF samples, such as the MAP2K1 c.371C>T mutation observed in 15 of the melanoma FFPE samples. The high concordance with digital PCR (Supplementary Section S6) generally suggests that these called variants are not due to systematic errors in the OCEANS method. A more likely explanation is that these mutations may have arisen from cytosine deamination DNA damage from FFPE treatment. This is supported by the fact that FFPE mutation calls had a significant over-representation of C>T and G>A mutations that derive from cytosine deamination. 

NGS VRF (%)

OCEANS VRF (%) 

Section S6: ddPCR Comparison Experiments

We performed a total of 24 ddPCR comparison experiments on 6 FFPE DNA samples, testing 4 mutations each (BRAF p.V600E, KRAS p.G13D, KRAS p.E62K, and MAP2K1 p.P124L). We observe that 21 of the 24 matched experiments gave concordant results, with 10 of these concordant results being concordant positives. 2 of the 11 concordant negative values were deemed OCEANS negative because they only satisfied one of the two criteria (based on VRF and Clair), suggesting that the orthogonal bioinformatic validation improved variant call accuracy. 
Individual ddPCR results and comparison to OCEANS % VRF are shown in figures S , S , S and S . Green dots are HEX (WT) positive droplets, blue dots are FAM (Variant) positive droplets, red dots are double positive, and black dots are negative droplets. VAF was calculated as VAF = ((Variant positive+Double positive)/(WT positive + Variant positive+Double positive)). 



  FIG. S1: Size verification of fragmented human genomic DNA. (a) 160 bp library run on a 2% agarose gel. (b) 10 kb library run on a 0.5% agarose gel. 


Fig. S shows

  Fig. S shows the design of SAL adapter sequences. Fig. S shows agarose gel electrophoresis analysis of SAL concatemer products before and after exonuclease digestion. The remaining bands after digestion are likely circular concatemer products. NS throughput for original amplicons vs. SAL products are shown in Fig. S . Fig. S shows the individual NS amplicon traces for 0% and 5% VAF samples shown in Fig. 2e. 




  FIG. S . Exonuclease treatment of SAL concatemers. 25 ng of SAL assembled concatemers of 100 bp DNA were treated with 10U of T7 exonuclease and 10U of Exonuclease VII to digest linear dsDNA. 




  Fig. S . Chromosome positions covered by the OCEANS panels are shown in 




  FIG. S . AML OCEANS panel performance without SAL. Comparison of variant read frequencies (VRF) observed for 7-plex AML OCEANS panel with or without SAL. The input samples used here were internal reference samples constructed by spiking known quantities of synthetic DNA bearing a mutation of interest into NA18562 gDNA at 1% VAF. No significant differences were observed in VRF values without SAL. However, the throughput for the MinION run without SAL was only 106 Mb in one hour compared to 620 Mb in one hour with SAL. 




  FIG. S1 . Scatter plot comparison of NS and Illumina NGS data on melanoma clinical samples. (a) Comparison on fresh frozen clinical samples. %VRF for each nucleotide position in the enrichment region is plotted against the corresponding %VRF in NGS. Number of data points in each quadrant is indicated. 8 putative variants in the top-left quadrant had both VRF >20% and Clair score above 180 (purple dots). 3 putative variants had either VRF >20% or Clair score above 180 (yellow dots). (b) ROC curve for fresh frozen clinical samples. NGS inferred VAF > 5% were considered true positives. (c) Comparison on FFPE clinical samples. VRF for each nucleotide position in the enrichment region is plotted against the corresponding VRF in NGS. Number of data points in each quadrant is indicated. 89 putative variants in the top-left quadrant had both VRF >20% and Clair score above 180 (purple dots). 54 putative variants had either VRF >20% or Clair score above 180 (yellow dots). (d) ROC curve for FFPE clinical samples. 




  FIG. S . NS and ddPCR comparison for mutation at BRAF (c.1799T) position in melanoma clinical samples. Top panel shows OCEANS results. Forward and reverse primer regions are shaded in gray, and the BDA enrichment region is shaded in green. Bottom panel shows ddPCR results for the same sample. 




  FIG. S . NS and ddPCR comparison for mutation at MAP2K1 (c.371C) position in melanoma clinical samples. Top panel shows OCEANS results. Forward and reverse primer regions are shaded in gray, and the BDA enrichment region is shaded in green. Bottom panel shows ddPCR results for the same sample. The c.371 position is indicated by arrow for FFPE sample 18. 


TABLE S1 .

 S1 S . Potential variants with both VRF> 20% and CLAIR score > 180 were definitively called as variants. Number of reads obtained and sampled for OCEANS melanoma panel, and the MNF for each sample. 


	CAATTCGGTCTCCCACTAGATGATGGGCTCCCGGAAGACAGTCCCCCAGGATGTTCGGATAGTTCCATTGGGACTTTCCACATCTTCT CACTCATCTGCAAAAACATCCCACGCCCC TAGTCCCTGGCTGGACCAAGCCCATCACCATTGGCAGGCACGCCCATGGCGACCAACACTGTGTTGAGATGGACCCCCTATTTATGGATTTATTTGTATTTGCCTTTAGCTAAATGTG TGTAAATACAGTTATACATATATGCATTCTCAATTTCATACCTTGCTTAATGAGGTGTAGATACCCAAAGATAAAGAATAAAAACACATACAAGTTGGAAATTTCTGGGCCATGAAAAA Sample NS reads NS throughput (Mb) Down-sampled reads MNF Sample SAL concatenated read (2925 nt) Synthetic 0% 40371 38.84 9948 22.66% NS concatenated reads Synthetic 0.05% 47480 40.24 10548 32.07% (491K reads) Synthetic 0.1% 46662 39.36 10361 35.05%
	AAAACATGCAAATCACATTATTGCCAACATGACTTGCCAGTCCCATAAGCATGACAACCTATGATGATAGGTTTACCCATCCTCAAAAGCCACTCATCTGCAAAAACATCCCACGCCT AGTCTAAGCTGCGTTGGCCCCATCACCGGTGGCAGGCACGCCCATGGCGACCAGCACTAGATGATGGGCTCCCGAAGTAGTCCCCCAGGGGATGTTGAATGGTTCCATTGGGACT TTCACATCTTCTCACTAGTTAGTTTTCACTACTACAAGTTAAAATGAATTTAAATAGTTTCTTTCTCCTCCAACTAATAGTGTATTCCACAGAGACAGCAGCCAGAAATATCCTCCTTAC TCATGGTCGGATCACAAGATTTGTGATTTTGGTCTATCAGACACATCAAGAATGATTCTAATTATGTGGATTTAAAGGACACTGAATGGGCTCCCAGAAGACAGTCCCCCAGGATGTT Synthetic 0.2% 48400 39.20 10916 41.17% Barcode demultiplex Synthetic 0.5% 49076 40.81 10920 45.14% with EPI2ME Synthetic 1.0% 38956 36.74 9777 48.71% CCAGATAGTTCCGTGGGACTTTTCCACATCTTCTCACTAGATGATGGGCTCCCGGAAGACAGTCCCCCAGGATGTTCAGATAGTTCCATTGGGACTTTTCCACATCTTCTCACTTGTG Horizon HD238 103621 89.98 11366 33.38% TTGAGATGGACAACCTATTTGTAAAGTTTATTTGTATTTGCCTTTAGCTAAATGTGTGTAAATATATACAGTTATACATATGCATTTCTCAATTTCATACCTTGCTTAATAGATTGTAGATAC CAAAGATAAGAATAAAACACATACAAGTTGGAAATTTCTGGGCCATAGAAAAAAAAAAAAAACATGCAAAATCACATTATTGCCAACATGACTTGCTTGATCCCCATAAGCATGACG Clinical FF2 106152 102.89 8305 33.25%
	ACCTATGATGATAGGTTTTACCCATCACTCACAAGCCACTAGATGGTGGGCTCCCGGTGGCATTTCTTGCCCGACCTGAGGATGTACCCGCCACGCCTGCAGGACTGACCTCTAGGT GGGCAAGCCGAGGCACAAGAGGGCGCGCTCTGGCGGAGTCAGCCCTCTTGTGAATTTGGGCTGGGAGACCAGCCAGGCCTAACAGGCAAGGCTGGGTCAGGCGAGGTCCTGG AGCCCACTTCGGTGTAGAGTGTGCAAAATGTGAACAGGCCTGCCCAGGATAGCTCCTGCATTAGCGCTGGTGCCTTACGAGCGGCCAGTCAGATTTTTATTTGGCACCACTACAGA GAGACCCAGGAGAGTCTCTTTAAGAAAATAGTTTAAACCACTAGATGATGGGCTCCCGGAAGACAGTCCCCCAGGATGTTCGGATAGTTCCGTGGGACTTTTCCATCTTCTCACTCC Other Clinical FF3 100151 103.39 8321 29.37% Library 1 121K reads Clinical FF20 101103 95.33 8601 41.76% libraries Clinical FF26 99803 91.88 7951 38.05% CGCCAGGAACGTGCTTGTCACCCACGGGAAAGTGGTGAGAATATGTGACTTTGGATTAGCTCGAGTTATCATGAGTGATTCCAACTATGTTGTCAGGGCACTCATCTGCAAAAACAT Clinical FF52 110598 103.63 8762 50.48% CCACACTAGTCCCTAGCTGGACCAAGCCCATCACCATTGGCAGGCACGCCCATGGCGACCAGCACTAGATGGGCTCCGGAAGACTGATCCCCAGGATGTTCCGGATAGTTCCATTG GGACTTTCCACATCTTCTCACTCCGCCAAGGAACGTGCCTTGTCACCCACGGGAAAGTGGTGAAGATATGTGACTTTGGTTGGCTCGAGTTATCATGAGTGATTCCAACTATGTTGT Clinical FF61 121686 106.37 9049 29.53%
	CAGGGCACTAGATGATGGGCTCCCCGGAAGACAGTCCCCAGGATGTTCCAGATAGTTCCATTGGGACTTTTCATCTTCTCACTAGATGATGGGCTCCCGGAAGACAGTCCCCCAGG Clinical FF172 134055 104.32 10165 38.43% ATGTTCCAGATAGTTCCATTGGGACTTTTCATCTTCTCACTAGAAGATTTCCTTGGAACTAAGCAGGCGTCAGAGGAGTTGGTGGGTGTGAGTGCCCTGTCCCTGCACTTCGGGTGG CTGCTGGTCCTCAGGTCCTGCTGTGTGGTTAGACGGCTTCCGGGCAGCCTGGTCTGGCCAACACTCACCTACCCTCTCTGCCTTTTCTCCCCAGAATTGTTGGTTTCCCAGTCCACTA TACTGACGTCTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTCACTTTAAAGTGTTTGGAATTAAATTACATCTAATTATAAAAATTTTCTTGGAGTCATATCTTTATCTAGAGTTA ACTCTCTGAGTGGTAGAATGAAAAAAACAGATGTTGAACTATGCAAGAGACATTTGAATTTATTGATGTCTATGAAGTGTTGTGGTTCCTTAGCCACATTTCTTTTTTTCAGGCTATTC AAGATCTCTGCATGGCAGTGGAGGAAGTCTCTTTAAAATAGTTTAAACCACTTTAAAGTGTTTGGACTAAATTACAAATACGAGTATAGGGTACTGCTTGGAGTCATATCTTTATCTAG AGTTAACTCTCTGGTGGTAGAATGAAAGTAGATATTGAACTATGCAAAGAGACATTTAATTTGATGTCTGTGAGTGTCATTCTGTTCATCTCCATCATGGCGGTGGAGGAAGTCTCTTT Clinical FFPE3 167541 119.61 8965 54.34% Extra Approx.10K Clinical FFPE4 131576 104.35 9066 61.42% reads Clinical FFPE5 124667 104.31 11127 63.51% concatenated reads Clinical FFPE7 152421 115.09 9986 60.51%
	AAGAAAATAGTTTAAACCACTAGATGATGGGCTCCCGGCCTCCTCTCCATTCAATGCCTGCCCCAACTCCCTGAGCTCTAGCTCCGCTGGTCTCCTCCGAGG Map amplicons with minimap2 and Clinical FFPE8 127153 96.82 9429 59.97%
	DNMT3A KIT NPM1 IDH1 IDH2-140 IDH2-172 FLT3 Off-target deconcatenate with python code Clinical FFPE10 115243 87.26 10381 62.78% Amplicon monomers: Clinical FFPE12 141370 110.07 10879 57.78%
	Mapped nucleotide fraction (MNF) = Off-target amplicons Clinical FFPE13 130721 Clinical FFPE14 157380 Clinical FFPE15 118950	[Length of Mapped (Underlined) reads] [Length of concatamer] 112.89 9565 51.72% x 100% On-target 119.88 9137 53.53% amplicons 105.75 9055 60.53%
	1711 nt 2925 nt 114.15 x 100% Align to GRCh38 reference 9999 49.36% Align to amplicon reference = Clinical FFPE17 179188 Clinical FFPE18 123029 92.40 10013 65.05% = 58.5% genome with minimap2 sequences with minimap2 Clinical FFPE19 160886 120.33 10643 58.53%
	Clinical FFPE20 178399	133.02	9844	58.3%
	Clinical FFPE21 135317	108.29	10147	58.42%
	Clinical FFPE23 118125 Alignment in BAM Clinical FFPE24 118941 format Clinical FFPE25 120325	99.96 97.34 108.32	Alignment in BAM format 8095 54.16% 9592 56.26% 9886 54.7%
		IGVtools			Downsample to coverage <150x with samtools
	% Variant base call			Downsampled BAM
		at each position			alignment file
			Clair DNMT3A IDH1 FLT3 IDH2 NPM1
	Positions with %VRF <20%	Subsample 1 Subsample 2 Positions Subsample 3 with Subsample 4 %VRF >20% Subsample 5	47.79 51.72 45.05 42.11 53.76	Variant calls with score >180 95.08 92.97 58.04 46.36 91.23 91.67 59.62 47.45 93.69 89.55 60.66 45.27 89.92 92.31 53.85 41.77 93.81 89.04 53.85 39.88	Variant calls with score <180
		Subsample 6	50.00	90.35 93.18 46.67 42.86
		Subsample 7 Subsample 8 Subsample 9	46.25 Overlapping 95.38 91.57 47.54 44.94 61.84 89.23 95.89 51.06 45.51 variant calls 42.22 93.58 95.79 56.60 41.90
		Subsample 10	47.78	93.86 92.00 57.69 41.13
		Subsample 11	41.56	92.54 93.24 49.09 41.21
		Subsample 12 Subsample 13	43.68 OCEANS 91.82 91.18 56.25 42.86 55.41 93.97 84.62 40.35 40.48
		Subsample 14 Subsample 15 Mean	48.86 variant 91.04 89.53 63.16 37.50 46.43 82.98 92.68 54.84 37.36 call 48.30 91.90 91.68 53.95 42.43
		Std.	5.60	3.10 2.79 6.08 3.04

FIG. S .Sample SAL concatenated read obtained on the OCEANS AML panel.Different colors show the 7 different amplicon sequences of the AML 7-plex panel.All the amplicons of the panel happen to be present in this concatenated read.Two off-target (brown) sequences are also present.CACT sequence (black) is the 4 nucleotide overhang sequence used in the SAL design.Underlined sequences are those that were mapped by minimap2 and deconcatenated by the custom python code.The deconcatenated MNF was calculated to be 58.5%.FIG.S .Schematic of bioinformatic workflow for OCEANS variant calling.TABLE S2. Variation in VRF (%) for different subsamples of 10,000 concatenated reads for the Horizon myeloid reference (HD829). 
Table S .

 S Table S to Table S . Spike-in mutations tested and their computed median enrichment fold (EF) values for AML and melanoma panel are shown in Table S 


	400					400			
					AKT1				PIK3CA-1047
	200 300 Clair Score				AKT3 BRAF KRAS-12 KRAS-61 MAP2K1-57 MAP2K1-121 MAP2K1-203	200 300 Clair Score			MAP2K2-125 MAP2K2-207 NRAS-61 NRAS-12 MAP2K2-57 PIK3CA-542
	100					100			
	0	0.05% 0.1%	0.2%	0.5%	1%	0	0.05% 0.1% 0.2%	0.5%	1%
			VAF				VAF		

FIG. S . Summary of Clair score for OCEANS melanoma panel. Mutations with Clair score > 180 are considered high confidence mutations. 
TABLE S .

 S Chromosome positions enriched by the 15-plex OCEANS melanoma panel. 


	Gene Enrichment Region (GRCh38)
	CTNNB1 Chr3: 41,224,607-41,224,626
	CTNNB1 Chr3: 41,224,645-41,224,663
	ARID1A Chr1: 26,729,717-26,729,730
	AXIN1	Chr16: 346,763-346,779
	TERT	Chr5: 1,295,114-1,295,123
	JAK1	Chr1: 64,845,513-64,845,528
	PTEN Chr10: 87,933,139-87,933,154
	TP53	Chr17: 7,675,081-7,675,091
	TP53	Chr17: 7,674,879-7,674,896
	TP53	Chr17: 7,674,210-7,674,222
	TP53	Chr17: 7,673,802-7,673,816

TABLE S .

 S Chromosome positions enriched by the 11-plex OCEANS HCC panel. Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing. BioRxiv, 2020.04.26.061747 (2020). 


	Gene	Enrichment Region (GRCh38)
	AKT1 Chr14: 104,780,200-104,780,218
	ALK	Chr2: 29,222,334-29,222,352
	ALK	Chr2: 29,220,830-29,220,847
	ALK	Chr2: 29,213,992-29,214,009
	ALK	Chr2: 29,209,816-29,209,832
	BRAF Chr7: 140,753,326-140,753,346
	BRAF Chr7: 140,781,595-140,781,614
	DDR2 Chr1: 162,778,599-162,778,613
	EGFR	Chr7: 55,174,001-55,174,015
	EGFR	Chr7: 55,174,769-55,174,790
	EGFR	Chr7: 55,181,309-55,181,322
	EGFR	Chr7: 55,181,378-55,181,391
	EGFR	Chr7

[1] Song, P., Chen, S. X., Yan, Y. H., Pinto, A., Cheng, L. Y., Dai, P., Patel, A. A., & Zhang, D. Y. 
TABLE S .

 S Chromosome positions enriched by the 28-plex OCEANS NSCLC panel. 


			,238
	TP53	Chr17: 7,675,081-7,675,091
	TP53	Chr17: 7,674,879-7,674,896
	TP53	Chr17: 7,674,217-7,674,230
	TP53	Chr17: 7,673,802-7,673,816
	Gene	Mutation	Median EF
	KIT	2446G>C	1420
	FLT3	2504A>T	886
	DNMT3A	2645G>A	32.4
	IDH1	394C>T	1280
	IDH2	418C>T	128
	IDH2	515G>T	61.7
	NPM1 863 864insTCTG	361
	TABLE S . Median Enrichment Fold (EF) values observed experimentally for the 7-plex AML OCEANS panel.
	Gene	Mutation	Median EF
	AKT1	235G>T	5650
	AKT3	49G>A	478
	BRAF	1799T>A	78.1
	KRAS	34G>A	625
	KRAS 180 181GA>TT	4220
	MAP2K1	169A>G	534
	MAP2K1	361A>T	221
	MAP2K1	607G>A	202
	MAP2K2	169T>G	811
	MAP2K2	373A>T	192
	MAP2K2	619G>A	2580
	NRAS	34G>A	143
	NRAS	181G>T	99.2
	PIK3CA	1624G>A	447
	PIK3CA	3139G>A	6570

TABLE S .

 S Median Enrichment Fold (EF) values observed experimentally for the 15-plex melanoma OCEANS panel. 


	Gene	Mutation	Median EF
	AKT1	49G>A	95
	ALK	3512T>A	168.6
	ALK	3520T>G	40.7
	ALK	3734T>G	63.5
	ALK	3806G>C	176
	BRAF	1799 1800delinsAT	296.2
	BRAF	1406G>T	15.6
	DDR2	2304T>A	105.4
	EGFR	2156G>C	11.8
	EGFR	2248G>C	21.2
	EGFR	2303G>T	39.4
	EGFR	2369C>T	154.3
	EGFR	2582T>A	46.3
	KRAS	183A>C	103.5
	KRAS	35G>A	81.7
	KRAS	436G>C	38.7
	MAP2K1	167A>C	13.6
	MET 3024 3028+7delAGAAGGTATATT	84.9
	NRAS	34G>A	85.9
	NRAS	181C>A	272.7
	PIK3CA	1624G>A	14.8
	PIK3CA	3140A>T	188
	PTEN	697C>T	180.2
	ROS1	5957C>T	178
	TP53	524G>A	82.6
	TP53	637C>T	17.3
	TP53	733G>A	135.2
	TP53	c.818G>A	587.2

TABLE S .

 S Median Enrichment Fold (EF) values observed experimentally for the 28-plex NSCLC OCEANS panel. 


	Gene	Mutation	Median EF
	CTNNB1	98C>G	93.4
	CTNNB1	133T>C	35
	ARID1A	1210C>T	49.5
	AXIN1	254G>A	770.9
	TERT promoter G>A	46.6
	JAK1	2108G>T	421.8
	PTEN	388C>A	855.7
	TP53	524G>A	179.8
	TP53	637C>T	409.2
	TP53	747G>T	51.3
	TP53	c.818G>A	693

TABLE S

 S 

. Median Enrichment Fold (EF) values observed experimentally for the 11-plex HCC OCEANS panel. 
TABLE S 3

 S . HCC clinical sample information. 



TABLE S .

 S Summary of ddPCR results for BRAF p.V600E, BRAF p.V600K, KRAS p.G13D, KRAS p.E62K, MAP2K1 p.P124L mutations in melanoma clinical samples. Concordant positive results are displayed in green, concordant negative results in blue, and discordant results in red. *OCEANS variant call satisfied one condition for a variant call, either VRF > 20% or Clair score >180. 


	Sample	BRAF p. V600	KRAS p. G13D	KRAS p. E62K	MAP2K1 p. P124L
		OCEANS ddPCR VAF OCEANS ddPCR VAF OCEANS ddPCR VAF OCEANS ddPCR VAF
	FFPE3	Yes	31.13%	Yes	0.03%	No	0%	Yes	0.28%
	FFPE13	Yes	0.66%	No*	0%	No	0%	Yes	0.15%
	FFPE17	No	0%	Yes	0%	No	0.43%	No	0%
	FFPE18	No	0%	Yes	0.04%	No	0%	No	0.50%
	FFPE19	No	0%	No*	0%	Yes	0.05%	Yes	0.64%
	FFPE25	No	0%	No	0%	Yes	0.02%	Yes	0.14%
	FF3	Yes	0.33%	NA	NA	NA	NA	NA	NA