www.sciencetranslationalmedicine.org/cgi/content/full/4/136/136ra68/DC1
`
`
`
`Supplementary Materials for
`
`
`
`Noninvasive Identification and Monitoring of Cancer Mutations by
`Targeted Deep Sequencing of Plasma DNA
`
`Tim Forshew, Muhammed Murtaza, Christine Parkinson, Davina Gale, Dana W. Y. Tsui,
`Fiona Kaper, Sarah-Jane Dawson, Anna M. Piskorz, Mercedes Jimenez-Linan, David
`Bentley, James Hadfield, Andrew P. May, Carlos Caldas, James D. Brenton,*
`Nitzan Rosenfeld*
`
`*To whom correspondence should be addressed. E-mail: nitzan.rosenfeld@cancer.org.uk (N.R.);
`james.brenton@cancer.org.uk (J.D.B.)
`
`Published 30 May 2012, Sci. Transl. Med. 4, 136ra68 (2012)
`DOI: 10.1126/scitranslmed.3003726
`
`
`The PDF file includes:
`
`
`Methods
`Fig. S1. PCR strategy and primer design.
`Fig. S2. Sanger traces for mutations identified by tagged-amplicon sequencing.
`Fig. S3. Background frequencies and detection limits for base substitutions.
`Fig. S4. Replicate dilute Sanger sequencing of a mutation identified in plasma.
`Table S1. Target-specific primers.
`Table S2. Unique sequencing barcodes.
`Table S3. Mutations identified in FFPE samples.
`Table S4. SNPs identified in circulating DNA from two plasma control samples.
`Table S5. Frequency of SNP alleles in dilution series of DNA from control
`plasma.
`Table S6. Additional data for Table 2 for mutations identified in plasma samples.
`Table S7. Mutations and amplicons studied in one breast cancer patient.
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`SUPPLEMENTARY METHODS
`
`Sanger sequencing
`Coding sequences of the TP53 gene (exons 2 to 11) were amplified as described previously
`(36) with the following modifications: PCR reactions were performed in 25 m
`l, universal
`primers M13 forward and M13 reverse were tagged onto both primers pairs and used to
`sequence in both the forward and reverse directions. To sequence exon 7, TP53-7F
`(CAGGTCTCCCCAAGGCGCAC) was used owing to a poly A tract downstream of the
`exon 7 forward primer described in (36): CATCCTGGCTAACGGTGAAAC. PCR products
`were sequenced on an ABI 3730 (Applied Biosystem). Mutational analysis was performed
`using Mutation Surveyor Software version 3.97 (SoftGenetics), using default settings
`(mutation score=5.00, mutation height=500, overlapping factor=0.20, dropping factor=0.20,
`SnRatio=1.00, mobility shift=imbedded algorithm). All exons were sequenced for 26
`samples. For 12 samples, sequencing was performed for selected exons only.
`
`Digital PCR
`The digital PCR method and the use of the BioMark system for digital PCR analysis have
`been described (7, 37). Samples were mixed with sequence-specific TaqMan probes, Master
`Mix, and 20× GE Sample Loading Reagent (Fluidigm). This mixture was then added to the
`12.765 Digital Array Chip (Fluidigm) where it was partitioned into 765 separate PCR
`reactions. These were PCR cycled on the Fluidigm BioMark and all positive reaction
`chambers counted. Amplifiable copies were calculated from the number of positive wells
`using a Poisson correction.
`
`Estimation of input DNA amounts
`DNA amount was calculated as total amplifiable copies, including both mutant and germline
`alleles, as determined by digital PCR (median amplicon size 84 bp, range 58-177 bp),
`multiplied by the estimated amount of DNA in 1 haploid genome (3.3 pg). Amplifiable
`copies in control healthy plasma samples were estimated using the FTH1 TaqMan assay
`Hs01694011_s1 (Life Technologies), which has an amplicon size of 180 bp.
`
`Dilution of plasma DNA samples to determine allele quantification accuracy
`After estimation of amplifiable copy numbers using digital PCR, both samples were diluted
`to 100 amplifiable copies per µl. A 5-µl mix was prepared containing 500 amplifiable copies,
`of which 400 copies were contributed by plasmaDna1 and 100 copies were contributed by
`plasmaDna2 (table s4). This was serially diluted 2.5× 6 times (reaching 244× dilution) in
`plasmaDna2. Each of the 7 mixes were then diluted 2.5× in water a further six times until we
`had an expected 2 total amplifiable copies of DNA. Twenty-eight of these mixes (table s5)
`were amplified using the described primer set (table S1) in triplicate, barcoded, and
`sequenced. Minor allele frequencies expected for the dilutions can be calculated using
`heterozygosity status for each SNP in either sample, an example that applies to 3 of the 5
`SNPs (rs1800899, rs1050171 and rs10241451) we studied is given in table s5.
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`Replicate dilute Sanger sequencing
`the primers EGFR_A_F
`We performed replicate dilute Sanger Sequencing using
`(CAGCAGGGTCTTCTCTGTTTCA) and EGFR_A_R (GGTGTTTTCACCAGTACGTTCCT).
`A PCR mastermix was made containing 0.05 U/µl FastStart High Fidelity Enzyme Blend
`(Roche), 1× FastStart High Fidelity Enzyme Buffer, 200 µM each of dNTPs, 4.5 mM MgCl2,
`5% DMSO, 50 nM of each primer, and 1X EvaGreen DNA binding dye (Biotium). Plasma
`DNA was quantified using digital PCR and diluted such that when partitioned in a 384-well
`plate, each reaction well would contain on average less than 1 amplifiable template. Forty-
`five cycles of PCR (50°C 2 min, 70°C 20 min, 95°C 10 min, 45 cycles of 95°C 15 s, 60°C 30
`s, 72°C 1 min) were performed on a 7900HT Fast Real-Time PCR machine (Applied
`Biosystems).
`
`
`
`TAm-Seq
`
`Primer design for TAm-Seq
`Target-specific primers were designed with universal primer sequences (termed CS1 and
`CS2) appended at the 5’-end (table S1). Primers were designed using Primer3 with a Tm
`range of 58-62°C, allowing homopolymer stretches no longer than 3 and keeping primer
`%GC less than 65%. Primer designs were screened against the hg19 reference genome to
`prevent cross-product generation, and were screened to prevent primer-dimer interactions and
`formation of intramolecular secondary structure. Primers were tested by amplifying from 10
`ng human genomic DNA (GM17317, Coriell) in 5 µl reaction volumes containing 0.05 U/µl
`FastStart High Fidelity Enzyme Blend (Roche), 1× FastStart High Fidelity Enzyme Buffer,
`200µM each of dNTPs, 4.5 mM MgCl2, 5% DMSO, 50 nM of each primer, and 1× Access
`Array sample loading solution (Fluidigm) using 35 cycles of amplification: 50°C 2 min, 70°C
`20 min, 95°C 10 min, 10 cycles of 95°C 15 s, 60°C 30 s, 72°C 60 s, 2 cycles of 95°C 15 s,
`80°C 30 s, 60°C 30 s, 72°C 60 s, 8 cycles of 95°C 15 s, 60°C 30 s, 72°C 60 s, 2 cycles of
`95°C 15 s, 80°C 30 s, 60°C 30 s, 72°C 60 s, 8 cycles of 95°C 15 s, 60°C 30 s, 72°C 60 s, 5
`cycles of 95°C 15 s, 80°C 30 s, 60°C 30 s, 72°C 60 s, 1 cycle of 72°C for 3 min.
`Barcode primers (table S2) comprised either the PE1 or PE2 sequences for Illumina
`cluster generation, a 10-bp barcode, followed by either CS1 or CS2 adaptor sequences. For
`example, one pair of barcode primers comprised 5’-PE1-CS1-3’+ 5’-PE2-BC-CS2-3’, the
`second pair comprised 5’-PE1-CS2-3’+ 5’-PE2-BC-CS1-3’.
`The sequencing reagent for read1 contained custom sequencing primers designed to
`anneal to both CS1 and CS2. The sequencing reagent for the index read contained custom
`sequencing primers designed to anneal to the reverse complements of CS1 and CS2.
`
`
`Preamplification for TAm-Seq
`Preamplification reactions were carried out in 10-µl reaction volumes containing 50 nM of
`each forward and reverse target-specific primer. Target DNA (1 to 5 µl) was added to a
`mastermix containing 0.5 U FastStart High Fidelity Enzyme Blend (Roche), 1× FastStart
`High Fidelity Enzyme Buffer, 200 µM each of dNTPs, 4.5 mM MgCl2, and 5% DMSO.
`Reactions were subjected to 15 cycles of amplification (95°C 10 min, 15 cycles of 95°C 15 s,
`60°C 4 min). Following preamplification, 4 µl Exo-SAP-it (Affymetrix) was added to each
`reaction and incubated for 15 min at 37°C, then for 15 min at 80°C. The preamplified
`samples were diluted 5-fold in PCR-grade water prior to amplification on the Access Array
`IFC system.
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`Target-specific amplification on the Access Array microfluidic system
`Individual primer pairs were loaded into the primer inlets of the Access Array IFC (Fluidigm)
`at a final concentration of 1 µM (20× the final concentration of 50 nM required for the PCR
`reaction) to account for the volume ratio of sample and primer chambers (20:1) in the Access
`Array IFC. One µl of each preamplified sample was added to 4 µl pre-sample mix containing
`0.05 U/µl FastStart High Fidelity Enzyme Blend (Roche), 1× FastStart High Fidelity Enzyme
`Buffer, 200 µM each of dNTPs, 4.5 mM MgCl2, 5% DMSO, and 1× Access Array sample
`loading solution (Fluidigm). Samples and primers were loaded into the IFC using an IFC-AX
`controller (Fluidigm). The volume of each sample chamber within the Integrated Fluidic
`Circuit (IFC) was 33 nl, containing 0.7% of the input reaction volume (5 µl). The IFC was
`then subjected to thermal cycling using a Biomark system (Fluidigm): 35 cycles of
`amplification (50°C 2 min, 70°C 20 min, 95°C 10 min, 10 cycles of 95°C 15 s, 60°C 30 s,
`72°C 60 s, 2 cycles of 95°C 15 s, 80°C 30 s, 60°C 30 s, 72°C 60 s, 8 cycles of 95°C 15 s,
`60°C 30 s, 72°C 60 s, 2 cycles of 95°C 15 s, 80°C 30 s, 60°C 30 s, 72°C 60 s, 8 cycles of
`95°C 15 s, 60°C 30 s, 72°C 60 s, 5 cycles of 95°C 15 s, 80°C 30 s, 60°C 30 s, 72°C 60 s, 1
`cycle of 72°C for 3 min). Harvesting solution (0.05% Tween-20) was loaded onto the IFC
`prior to harvesting on an IFC-AX controller. One µl of harvested product was then
`transferred to a clean PCR plate containing 99 µl PCR-free water.
`
`
`
`Sequencing adaptor and barcode primer addition
`For each sample, 1 µl of the 100-fold diluted PCR products was added to each of two PCR
`plates containing 15 µl pre-sample mastermix containing 0.05 U/µl FastStart High Fidelity
`Enzyme Blend (Roche), 1× FastStart High Fidelity Enzyme Buffer, 200 µM each of dNTPs,
`4.5 mM MgCl2, and 5% DMSO. In the first plate, 4 µl of one pair of primers containing an
`individual 10-base barcode (BC) sequence, and sequence tags for reading in one direction
`(PE1-BC-CS1 + PE2-CS2) were added to each well. In the second plate, 4 µl of primers
`containing (PE1-BC-CS2 + PE2-CS1) were added to each well. The corresponding wells in
`both plates contained primers with the same barcode sequence (e.g. plate 1, well A1 =
`Barcode FLD0001, plate 2, well A1 = barcode FLD0001). Reaction products in plates were
`amplified for 15 cycles: 95°C 10 min, 15 cycles of 95°C 15 s, 60°C 30 s, 72°C 4 min, 1 cycle
`of 72°C for 3 min.
`
`
`
`Quantification and clean up of DNA library
`After PCR products were barcoded, they were analyzed using Agilent 2100 BioAnalyzer to
`ensure expected insert size (~180 bp) was obtained. They were then pooled together and
`purified using AMPure XP beads using a bead to amplicon ratio of 1.8:1. The library was
`quantified by Agilent BioAnalyzer and subjected to Illumina cluster generation. Single-end
`sequencing of 100 bases was performed on an Illumina GAIIx sequencer followed by a 10-
`base indexing (barcode) read, using custom sequencing primers targeted to the CS1 and CS2
`tags for both read1 and the index read according to manufacturer’s recommendations.
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`Analysis of sequencing data
`De-multiplexing and alignment. Reads generated by the primary Illumina pipeline
`were demultiplexed using a known list of barcodes (table S2), allowing one base mismatch
`out of 10 bases. Each set of reads was aligned independently to the hg19 reference genome
`using bwa-short in the single-end mode. Using expected genomic positions, each set of
`aligned reads was separated further into its constituent amplicons. A pileup was generated for
`each amplicon using samtools v1.12a. Using a base quality and a mapping quality cut-off of
`30, observed frequencies of non-reference alleles for every sequenced locus across all
`amplicons and barcodes were calculated.
`Mutation identification. For each locus and base, the distribution of non-reference
`background allele frequencies/reads was modeled as a Normal distribution and as a Poisson
`distribution, fitting to a Normal distribution and using the mean number of observed non-
`reference reads in a set of representative barcodes as the parameter for the Poisson
`distribution. For each barcode, the probability of obtaining the observed frequency/number of
`reads (or greater) was calculated in both models, and the lower value was retained. Putative
`substitutions that passed a probability cut-off (confidence margin) of 0.9995 were kept for
`further analysis as candidate mutations.
`For each barcode, all loci that passed the probability cut-off were ranked by observed
`frequency, corrected for the median frequency (over all samples) for each locus/base. Known
`SNPs obtained from the 1000 Genomes project and regions covering amplification primers
`were discarded, leaving 17,934 possible base calls per sample. Indels are not analyzed in the
`current calling algorithm. A point mutation was called in a sample if it ranked among the 6
`loci with highest non-reference frequency in both duplicates, and was represented by at least
`10 reads in each. If a mutation was called, it was also discarded from the pool and the process
`was repeated by testing the 6 highest remaining frequencies. Mutations that occurred at low
`frequency may have fallen outside the list of top-ranked loci, especially for more noisy
`samples, and may have therefore been missed. For each sample, we estimated the threshold
`for mutation calling by the highest background-corrected non-reference allele frequency that
`did not get into the top-ranked list and may have been missed by the calling algorithm. For
`mutations called
`in multiple (overlapping) amplicons,
`the amplicon showing best
`concordance between duplicates was retained.
`For the sequencing data obtained from a mix of FFPE samples, we performed an
`additional lower-stringency analysis using a probability cut-off of 0.9995 keeping the 12 loci
`with highest non-reference frequency to enhance sensitivity. For the data obtained from
`plasma sequencing, we performed an additional higher-stringency analysis using a
`probability cut-off of 0.9999 keeping the 6 loci with highest non-reference frequency to
`enhance sensitivity.
`Mutation detection. When applying TAm-Seq to measure a pre-defined mutation (as
`opposed to screening nearly 18,000 possible substitutions), the frequency of the mutant allele
`can be read out directly from the data at the desired locus. We used confidence margins of
`~0.95 using a Normal distribution model for each examined substitution, and requiring a
`minimum of 10 representative reads, in at least one amplified library per sample. When a
`mutation was positively detected, its allele frequency was estimated by averaging the allele
`frequencies in all replicates after subtracting substitution-specific background frequencies.
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` SUPPLEMENTARY FIGURES
`
`
`
`
`
`
`
`
`
`
`
`
`Figure S1: PCR strategy and primer design. Target-specific primers were synthesised with
`common adaptor sequences at their 5’ ends (CS1 and CS2). These were used during the
`preamplification and single-plex stages to amplify the selected regions. Sequencing platform-
`specific adaptors and unique sample barcodes were attached during an additional round of
`PCR. The barcoding primers consist of Illumina sequencer adaptors (PE1 and PE2) at their 3’
`end, a unique 10 base barcode, and the common CS1 and CS2. Permutations of the universal
`tags and the sequencing adaptors enabled construction of bidirectional barcoded sequencing
`libraries. Using custom sequencing primers directed to the two tag sequences, sequence reads
`representing both strands of the amplicon pool were obtained from a single-end run. Both
`combinations were used in order to barcode and read amplicons in both directions. All
`samples were then pooled, cleaned, and then sequenced.
`
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`A
`
`B
`
`
`
`
`
`
`
`Figure S2: Sanger traces for mutations identified by tagged-amplicon sequencing.
`Mutational analysis of Sanger traces was performed by Mutation Surveyor Software version
`3.97 (SoftGenetics) using default settings. The TP53 gene was on the reverse strand,
`therefore mutations seen in the sequencing traces were the reverse complement of the
`mutations as indicated by the hg19 coordinates. (A) Sanger sequencing of sample #104.
`Highlighted locus shows a mutation that was identified by TAm-Seq (17:7578370C>T), but
`not Sanger sequencing. The Sanger sequencing trace for the sample (middle panel) shows a
`weak signal above the reference (top panel), but this was at background noise levels and did
`not pass detection thresholds (lower panel). (B) Sanger sequencing of sample #111.
`Highlighted locus shows a mutation that was identified by TAm-Seq (17:7579642C>T), but
`not Sanger sequencing. The Sanger sequencing trace (lower panel) showed a weak signal
`above the reference (top panel), but this did not pass detection thresholds (middle panel).
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`Figure S3: Background frequencies and detection limits for base substitutions. For each
`one of the possible base substitutions in the sequenced amplicons, we calculated the mean
`background read rate in the 124 barcoded libraries generated from the set of 62 plasma
`samples (cumulative distribution shown in black), and the standard deviation. Colored lines
`show the cumulative distribution of frequencies that would exceed the indicated confidence
`margins, using a Normal distribution model for each base substitution, given its measured
`mean and standard deviation. Substitutions that were covered by more than one amplicon or
`read direction were included multiple times, once for each amplicon/direction in which they
`were sequenced. Substitutions that had zero reads in all barcodes had undefined confidence
`limits and were omitted (n = 589). Dashed grey lines indicate the median value for 0.99
`confidence margin.
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`E1
`
`A1
`
`
`
`
`
`
`
`H23
`
`H5
`
`
`
`
`
`
`
`
`
`
`
`Figure S4: Replicate dilute Sanger sequencing of a mutation identified in plasma.
`Replicate Sanger sequencing of highly dilute template was used to validate the EGFR
`mutation that was identified de novo in plasma and was not found in the corresponding
`tumour sample from the same patient (Patient #27, Table 1). The panels show examples of
`mutant and wild type reads in either direction, with the mutated base (chr7:55259437G>A,
`genome build hg19 coordinates) highlighted in blue. E1 is an example of mutant forward
`read, and A1 is a matched wild-type. H23 is an example of mutant reverse read, and H5 a
`matched wild-type.
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`SUPPLEMENTARY TABLES
`
`
`Table S1: Target-specific primers. Each target-specific primer consists of a universal 5’ end and a target-specific 3’ end as follows:
`
`5’-ACACTGACGACATGGTTCTACA-[Target Specific-Forward]-3’.
`
`5’-TACGGTAGCAGAGACTTGGTCT-[Target Specific-Reverse]-3’
`
`
`
`Gene name and Amplicon ID Target Specific Primer – Forward
`
`Target Specific Primer- Reverse
`
`Chr Amplicon Start Amplicon
`End
`
`PIK3CA_E00001077674 *
`PIK3CA_E00001139987
`EGFR_Exon19
`EGFR_E00001601336_1
`EGFR_E00001601336_2
`EGFR_E00001681524_1
`EGFR_E00001681524_2
`EGFR_E00001631695_1
`EGFR_E00001779947_1
`EGFR_E00001779947_2
`EGFR_E00001790701_1
`EGFR_E00001801208_1
`EGFR_E00001801208_2
`EGFR_E00001773562_1
`BRAF_E00002324725
`PTEN_E00001456562_1
`PTEN_E00001156351_1
`PTEN_E00001156344_1
`PTEN_E00001156337_4
`PTEN_E00001156330_1
`
`
`
`AACAGAGAATCTCCATTTTAGCAC
`CAGAGGGGAAAAATATGACAAA
`GGTCTTTGCCTGCTGAGAGT
`TGAGCAAGAGGCTTTGGAGT
`CCACACAGCAAAGCAGAAAC
`TCACAATTGCCAGTTAACGTCT
`CCGGACATAGTCCAGGAGG
`GCGTCTTCACCTGGAAGGG
`GGCTCCTTATCTCCCCTCC
`GCGTGGACAACCCCCAC
`TTCTCTTCCGCACCCAG
`GGATGCAGAGCTTCTTCCCA
`GCTGACCTAAAGCCACCTCC
`GGTCTTCTCTGTTTCAGGGCAT
`GGCCTCAGTACAAACTCATTAGC
`GTGTCACTCGTAATTAGGTCCA
`CCACCAGTCACTCACACTTG
`TGTTCATTCATGATCCCACTGC
`AGGGATGCAAAGGCCTCA
`TCCCTGCCAGCGAGAT
`CAATGGAAGCACAGACTGCAA
`GCCTTCTTTAAGCAATGCCATCTTTAT
`ATGAGGTACTCGTCGGCATC
`CCCCTGCTCCTATAGCCAA
`GTTCAAATGAGTAGACACAGCTT
`ACTTCTACCGTGCCCTGA
`GGAGAGCTGTAAATTCTGGCTT
`TACCCTCCATGAGGCACAC
`CTGATGGGACCCACTCCAT
`TCATAATGCTTGCTCTGATAGGA
`TCCGTCTACTCCCACGTTCT
`GCAGCTTCTGCCATCTCTCT
`ATGAAAACACAACATGAATATAAACATCAAT
`TGCTGCATATTTCAGATATTTCTTTCCTTA
`AATAGTTGTTTTAGAAGATATTTGCAAGC
`AATCTGTCTTTTGGTTTTTCTTGATAGT
`TATATCACTTTTAAACTTTTCTTTTAGTTGTGC CTCGATAATCTGGATGACTCATTATTGTT
`TTCTTATTCTGAGGTTATCTTTTTACCAC
`TCATTACACCAGTTCGTCCCT
`
`3
`3
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`7
`10
`10
`10
`10
`10
`
`178935943 178936150
`178952038 178952227
`55242373
`55242537
`55248902
`55249111
`55249005
`55249213
`55259352
`55259542
`55259395
`55259591
`55260366
`55260575
`55266362
`55266571
`55266461
`55266641
`55267932
`55268134
`55268806
`55268987
`55268921
`55269101
`55269336
`55269516
`140453108 140453256
`89624175
`89624372
`89653738
`89653927
`89685172
`89685367
`89690776
`89690940
`89692739
`89692919
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`PTEN_E00001156330_3
`PTEN_E00001156327_1
`PTEN_E00001156327_4
`PTEN_E00001156321_1
`PTEN_E00001156321_2
`PTEN_E00001156321_4m
`PTEN_E00001156315_1m
`PTEN_E00001156315_5 *
`PTEN_E00001156315_7
`PTEN_E00001456541_1
`PTEN_E00001456541_2 *
`KRAS_E00000936617
`TP53_E00001757276_1
`TP53_E00001728015_1
`TP53_00001404886_13
`TP53_E00001789298_1
`TP53_E00001789298_2
`TP53_E00001789298_3
`TP53_E00001665758_1
`TP53_E00001255919_1
`TP53_E00001255919_3
`TP53_E00001255919_5
`TP53_E00001255919_6
`TP53_E00001612188_1
`TP53_E00001612188_2
`TP53_E00002359670
`TP53_E00002419584
`TP53_E00001596491_1
`
`*Each of these 3 primer pairs potentially amplifies an additional non-target region, owing to large regions of homology and the short amplicon
`size. This was taken into account in data analysis.
`
`TGACCAATGGCTAAGTGAAGATGA
`TCTTAAATGGCTACGACCCAG
`CAGTCAGAGGCGCTATGTGT
`TGACAGTTTGACAGTTAAAGGCAT
`TGTGGTCTGCCAGCTAAAGG
`TCCACAAACAGAACAAGATGCT
`GCAACAGATAACTCAGATTGCCTT
`AGGACAAAATGTTTCACTTTTGGGTAA
`CCTCAGAAAAAGTAGAAAATGGAAGTC
`AGATGAGTCATATTTGTGGGTTTTCA
`GTAGAGGAGCCGTCAAATCCA
`GCCTGCTGAAAATGACTGAA
`GACCCAAAACCCAAAATGGC
`GGAATCCTATGGCTTTCCAACC
`TCTGTATCAGGCAAAGTCATAGAA
`AGAAAACGGCATTTTGAGTGT
`CTGGTGTTGTTGGGCAGT
`TGTCCTGCTTGCTTACCTCG
`GGGGTCAGAGGCAAGCAG
`GAGAAAGCCCCCCTACTGC
`TCCAAATACTCCACACGCAAA
`AGCTGCTCACCATCGCTA
`TGTGCTGTGACTGCTTGTAG
`ATACGGCCAGGCATTGAAGT
`GGAAACCGTAGCTGCCCTG
`CAGCCTCTGGCATTCTGG
`TCAAATCATCCATTGCTTGG
`TTTCGCTTCCCACAGGTCTC
`
`TCCAGGAAGAGGAAAGGAAAAACA
`TCCAGATGATTCTTTAACAGGTAGC
`TCTAGATATGGTTAAGAAAACTGTTCCA
`CACACACAGGTAACGGCTGA
`TCTCCCAATGAAAGTAAAGTACAAACC
`GGCCTTTTCCTTCAAACAGGATT
`GTTTCCTCTGGTCCTGGTATGA
`ACTAGATATTCCTTGTCATTATCTGCAC
`ACAAGTCAACAACCCCCACA
`TCTGGATCAGAGTCAGTGGT
`TTCATGGTGTTTTATCCCTCTTGA
`AGAATGGTCCTGCACCAGTAA
`TCCCTGCTTCTGTCTCCTAC
`CCCCCTCCTCTGTTGCTG
`GCCTCAAAGACAATGGCTCC
`AAGGGTGCAGTTATGCCTCA
`ATCTCCGCAAGAAAGGGGAG
`GCCTCTTGCTTCTCTTTTCCT
`CTTGGGCCTGTGTTATCTCC
`AGCATCTTATCCGAGTGGAAGG
`GCTGCCCCCACCATGAG
`CCAACTGGCCAAGACCT
`TGCCCTGACTTTCAACTCTGT
`CCTCCTGGCCCCTGTC
`AAGACCCAGGTCCAGATGAA
`CCTGGTCCTCTGACTGCTCT
`CCATGGGACTGACTTTCTGC
`CAGCCAGACTGCCTTCCG
`
`10
`10
`10
`10
`10
`10
`10
`10
`10
`10
`10
`12
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`17
`
`89692840
`89711775
`89711889
`89717547
`89717620
`89717748
`89720457
`89720649
`89720706
`89724997
`89725068
`25398163
`7572850
`7573859
`7576584
`7576786
`7576908
`7577003
`7577432
`7578091
`7578229
`7578361
`7578425
`7579260
`7579359
`7579479
`7579557
`7579758
`
`89693048
`89711942
`89712077
`89717726
`89717802
`89717956
`89720706
`89720799
`89720915
`89725180
`89725264
`25398329
`7573030
`7574054
`7576734
`7576983
`7577075
`7577187
`7577631
`7578274
`7578406
`7578525
`7578594
`7579421
`7579520
`7579626
`7579754
`7579940
`
`
`
`10
`
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`
`

`

`Table S2: Unique sequencing barcodes. Platform-specific adaptors and barcodes are
`attached through PCR following the single-plex amplification step. The primers consisted of
`the PE1 and PE2 sequences for Illumina cluster generation, a 10-bp barcode, and the CS1 and
`CS2 adaptors, used in pairs: PE1-CS1 with PE2-BC-CS2, and PE1-CS2 with PE2-BC-CS1.
`PE1: AATGATACGGCGACCACCGAGATCT.
`PE2: CAAGCAGAAGACGGCATACGAGAT.
`
`BarcodeBarcodeBarcode BarcodeBarcode BarcodeBarcodeBarcode
`
`
`
`
`
`
`nnnnameameameame
`
`
`
`ssssequenceequenceequenceequence
`FLD0001 GTATCGTCGT
`FLD0002 GTGTATGCGT
`FLD0003 TGCTCGTAGT
`FLD0004 GTCGTCGTCT
`FLD0005 GTGCGTGTGT
`FLD0006 GCGTCGTGTA
`FLD0007 GTCGTGTACT
`FLD0008 GATGTAGCGT
`FLD0009 GAGTGATCGT
`FLD0010 CGCTATCAGT
`FLD0011 CGCTGTAGTC
`FLD0012 GCTAGTGAGT
`FLD0013 GAGCTAGTGA
`FLD0014 CGTGCTGTCA
`FLD0015 GATCGTCTCT
`FLD0016 GTGCTGTCGT
`FLD0017 TGAGCGTGCT
`FLD0018 CATGTCGTCA
`FLD0019 TCAGTGTCTC
`FLD0020 GTGCTCATGT
`FLD0021 CGTATCTCGA
`FLD0022 GTCATGCGTC
`FLD0023 CTATGCGATC
`FLD0024 TGCTATGCTG
`FLD0025 TGTGTGCATG
`FLD0026 GAGTGTCACT
`FLD0027 CTAGTCTCGT
`FLD0028 GAGTGCATCT
`FLD0029 TGCGTAGTCG
`FLD0030 CTGTGTCGTC
`FLD0031 CTGTAGTGCG
`FLD0032 GTGCGCTAGT
`FLD0033 TGTGCTCGCA
`FLD0034 GATGCGAGCT
`FLD0035 CTGTACGTGA
`FLD0036 GCGATGATGA
`FLD0037 TGTCGAGTCA
`FLD0038 GTCTACTGTC
`FLD0039 CAGTCAGAGT
`FLD0040 CGCAGTCTAT
`FLD0041 GTATGAGCAC
`FLD0042 CGAGTGCTGT
`FLD0043 TATAGCACGC
`FLD0044 TCATGCGCGA
`FLD0045 TATGCGCTGC
`FLD0046 TCTCTGTGCA
`FLD0047 CTATCGCGTG
`FLD0048 TACGCTGCTG
`FLD0049 CTGCATGATC
`FLD0050 CGCGTATCAT
`FLD0051 GTATCTCTCG
`FLD0052 GCTCATATGC
`FLD0053 CACTATGTCG
`FLD0054 TAGCGCGTAG
`FLD0055 CGTCACAGTA
`FLD0056 TCGCGTGAGA
`FLD0057 TACATCGCTG
`FLD0058 GTGAGAGACA
`FLD0059 GACTGTACGT
`
`FLD0121 CAGAGCTAGT
`FLD0122 CGCAGAGCAT
`FLD0123 TGTACAGCGA
`FLD0124 ACGTCAGTAT
`FLD0125 TCACAGCATA
`FLD0126 ACTGCGTGTC
`FLD0127 CGATCGACTG
`FLD0128 GCGAGATGTA
`FLD0129 CTGATGCAGA
`FLD0130 GTGACGTACG
`FLD0131 CGACGCTGAT
`FLD0132 CTACGATCAG
`FLD0133 GCACTAGACA
`FLD0134 CTAGCAGATG
`FLD0135 CATGATACGC
`FLD0136 GCAGCTGTCA
`FLD0137 ACGTATCATC
`FLD0138 AGTATCGTAC
`FLD0139 GATACACTGA
`FLD0140 GACTAGTCAG
`FLD0141 GATGACTACG
`FLD0142 CAGAGAGTCA
`FLD0143 TCGATCGACA
`FLD0144 ACTGATGTAG
`FLD0145 ACTCGATAGT
`FLD0146 GACGATCGCA
`FLD0147 TCATCATGCG
`FLD0148 ACATGTCTGA
`FLD0149 AGTCATCGCA
`FLD0150 TAGCATACAG
`FLD0151 AGAGTCGCGT
`FLD0152 TCTACGACAT
`FLD0153 CACGAGATGA
`FLD0154 ACGCACATAT
`FLD0155 ACGTGCTCTG
`FLD0156 ACGATCACAT
`FLD0157 AGTGTACTCA
`FLD0158 TGATGTATGT
`FLD0159 GATATATGTC
`FLD0160 TAGTACTAGA
`FLD0161 TATAGAGATC
`FLD0162 TCGATATCTA
`FLD0163 TACATGATAG
`FLD0164 TGAGATCATA
`FLD0165 CTACATACTA
`FLD0166 ATCAGTGTAT
`FLD0167 ATCATATCTC
`FLD0168 AGTAGATCAT
`FLD0169 ACATAGTATC
`FLD0170 ATGTATAGTC
`FLD0171 ACAGTCATAT
`FLD0172 ACATATACGT
`FLD0173 AGCATCTATA
`FLD0174 AGACTATATC
`FLD0175 CAGCATCTAG
`FLD0176 CGAGACGACA
`FLD0177 ATCACTCATA
`FLD0178 AGCTCTGTGA
`FLD0179 ATGTCATGCT
`FLD0180 GCTGACAGAG
`FLD0181 ATACAGTCTC
`
`FLD0060 GCACGTAGCT
`FLD0061 TCACGCTATG
`FLD0062 CGTACTACGT
`FLD0063 CAGCTGAGTA
`FLD0064 GAGATCAGTC
`FLD0065 TACTGAGCTG
`FLD0066 TAGTAGCGCG
`FLD0067 GACGTCTGCT
`FLD0068 GTACTCGCGA
`FLD0069 TCTGAGCGCA
`FLD0070 TAGACGTGCT
`FLD0071 GTGACTCGTC
`FLD0072 TCGAGTAGCG
`FLD0073 CGTATGATGT
`FLD0074 TAGTCTGTCA
`FLD0075 TGTCTCTATC
`FLD0076 CTAGAGTATC
`FLD0077 TATCATGTGC
`FLD0078 CATGAGTGTA
`FLD0079 TGTCGTCATA
`FLD0080 TATCTCATGC
`FLD0081 TGTGTCACTA
`FLD0082 TATCGATGCT
`FLD0083 TAGAGTCTGT
`FLD0084 CATGCATCAT
`FLD0085 TGATCAGTCA
`FLD0086 CGTCTATGAT
`FLD0087 GTGATACTGA
`FLD0088 CTAGATCTGA
`FLD0089 TATCAGTCTG
`FLD0090 TCAGATGCTA
`FLD0091 TATGTACGTG
`FLD0092 CTATACAGTG
`FLD0093 TGATACTCTG
`FLD0094 TCAGCGATAT
`FLD0095 CTACTGATGA
`FLD0096 GTAGTACACA
`FLD0097 TGCTACATCA
`FLD0098 AGTGTGTCTA
`FLD0099 TCATATCGCG
`FLD0100 TACGTATAGC
`FLD0101 CAGCTATAGC
`FLD0102 TCGATGCGCT
`FLD0103 GCACGCGTAT
`FLD0104 GCAGTATGCG
`FLD0105 TGATAGAGAG
`FLD0106 GCTACTAGCG
`FLD0107 TGCGAGACGT
`FLD0108 CGATGACAGA
`FLD0109 GACTCATGCT
`FLD0110 GTCTGATACG
`FLD0111 ACTAGCTGTC
`FLD0112 GCGTAGACGA
`FLD0113 CTCAGCAGTG
`FLD0114 CAGTCTACAT
`FLD0115 TACTGCAGCG
`FLD0116 TACACAGTAG
`FLD0117 CACATACAGT
`FLD0118 CACAGTGATG
`FLD0119 CGAGCTAGCA
`FLD0120 GAGACTATGC
`
`
`
`11
`
`Foresight EX1032
`Foresight v Personalis
`
`

`

`FLD0182 CATAGACGTG
`FLD0183 AGAGATATCA
`FLD0184 ATGCTGCGCT
`FLD0185 AGTCAGACGC
`FLD0186 ACGATACACT
`FLD0187 AGCGAGTATG
`FLD0188 ATCGCTACAT
`FLD0189 ATGCTAGAGA
`FLD0190 AGCAGTACTC
`FLD0191 ATCTAGATCA
`FLD0192 ATCGCATAGA
`FLD0193 TTGTTGCTGT
`FLD0194 GTGTGGTTGT
`FLD0195 TAGGTGGAAT
`FLD0196 TGTAGGTGGA
`FLD0197 TTAGTGGTGA
`FLD0198 GTGAAGGTAA
`FLD0199 TGTTGTGGTA
`FLD0200 GTTGATGAGT
`FLD0201 GGTCAGTGTA
`FLD0202 GTAATGGAGT
`FLD0203 CTCGTTATTC
`FLD0204 GGAAGTAAGG
`FLD0205 CGGTGTGTGT
`FLD0206 CGTCTTCTTA
`FLD0207 TGTGAATCTC
`FLD0208 CTAATCGTGT
`FLD0209 CTCTTAGTTC
`FLD0210 GGATAGGATC
`FLD0211 GGTGTCTTGT
`FLD0212 GATGGTTGTA
`FLD0213 CCTCGTTGTT
`FLD0214 GGTTGGAGTT
`FLD0215 TGGTGTCCGT
`FLD0216 CGTTAGCGTA
`FLD0217 TACTAGGATC
`FLD0218 GTCTCAATGT
`FLD0219 GATGAGGTAT
`FLD0220 GGTGTTAGTG
`FLD0221 CATTCTCTGA
`FLD0222 CATCTGGAGT
`FLD0223 GAATGGAAGA
`FLD0224 GGCTGTGATC
`FLD0225 TGGTGCTGGA
`FLD0226 TATGGTAAGG
`FLD0227 GTTCGATTGT
`FLD0228 GGTAGAATGA
`FLD0229 TTCTCATCGT
`FLD0230 CTCAATCGTA
`FLD0231 CGCTAATGTA
`FLD0232 GCGTCTGAAT
`FLD0233 TTCTGTTGCC
`FLD0234 TTGTCCTTGC
`FLD0235 CCTGTGTAGA
`FLD0236 GATAAGAAGG
`FLD0237 CAGGTCACAT
`FLD0238 GCCATGTCAT
`FLD0239 TCTGCCTATA
`FLD0240 CTTAGTTCGC
`FLD0241 CGTAATGAGC
`FLD0242 TTGCTTAGTC
`FLD0243 TCTTGTTCAC
`FLD0244 GTGGCTTCGT
`FLD0245 TGTTCGATAG
`FLD0246 TCATTCAGTG
`FLD0247 GTGGAGAGCT
`FLD0248 GTAGAAGTGG
`FLD0249 TGGAGCATGT
`
`FLD0250 GAAGGAGATA
`FLD0251 CGAATGTATG
`FLD0252 TCGTGAATGA
`FLD0253 GAATAGCTGA
`FLD0254 TTGTCACATC
`FLD0255 CTGGAGGCTA
`FLD0256 TGTCAGCTTA
`FLD0257 GTTCTTCGTA
`FLD0258 TTACACGTTC
`FLD0259 GTAGCCAGTA
`FLD0260 TGAGAAGGTA
`FLD0261 CCATATGATC
`FLD0262 CGATCCTATA
`FLD0263 TGACTAGCTT
`FLD0264 TAACTCTGCT
`FLD0265 TCGAATGTGC
`FLD0266 TCGCTGAACA
`FLD0267 GCGTTATTGC
`FLD0268 GAACTATCAC
`FLD0269 TCGAGGTACT
`FLD0270 TGCGGATGGT
`FLD0271 TTCGAGCTAT
`FLD0272 GGTCTGGTGT
`FLD0273 CTAAGTCATG
`FLD0274 TTGCAGATCA
`FLD0275 CTGCGAATGT
`FLD0276 CTGTTCTAGC
`FLD0277 CACTTGTGTG
`FLD0278 TGGATGACAT
`FLD0279 GATCCTGAGC
`FLD0280 GTCGGTCTGA
`FLD0281 TGTTACGATC
`FLD0282 GTCTTGGCTC
`FLD0283 GGTCGTGCAT
`FLD0284 CAGGCTCAGT
`FLD0285 TAGCTTCACT
`FLD0286 CAGATGTCCT
`FLD0287 TTACGCAGTG
`FLD0288 TTCGTTCCTG
`FLD0289 CACTGCTTGA
`FLD0290 TCTAGCGTGG
`FLD0291 GCATAATCGC
`FLD0292 GTCGTAACAC
`FLD0293 GAGATTGCTA
`FLD0294 GGACAGATGG
`FLD0295 CTTACGTTGC
`FLD0296 GTGTTCGGTC
`FLD0297 CTCAAGAAGC
`FLD0298 TCTCGGATAG
`FLD0299 CTCTGGACGA
`FLD0300 CGAGCATTGT
`FLD0301 CCAAGAAGAA
`FLD0302 TCCTTGTTCT
`FLD0303 GTAACGATGT
`FLD0304 TGGACTCAGA
`FLD0305 GGCATCATGC
`FLD0306 GTATAACGCT
`FLD0307 GCAGATAAGT
`FLD0308 GTCGGCTCTA
`FLD0309 TTCGATAGCA
`FLD0310 GTCTAGCAGG
`FLD0311 GGAACACAGG
`FLD0312 TGGTTCGCTG
`FLD0313 CACATTAGCG
`FLD0314 GAAGCGCACT
`FLD0315 GCATGCCAGT
`FLD0316 GGAGACTGTA
`FLD0317 TCGAACTGCA
`
`FLD0318 GAGAGGACAT
`FLD0319 GAGCACGGAA
`FLD0320 GCTCTAACAT
`FLD0321 TGCTGGCTTG
`FLD0322 TGCATGGAGC
`FLD0323 GTACTAAGAG
`FLD0324 GAAGTCAAGC
`FLD0325 GCGCATTATG
`FLD0326 GTCCAGACAT
`FLD0327 GAGACCTCTA
`FLD0328 TTGCACTCAG
`FLD0329 TGCGGCGATA
`FLD0330 AGTTGCTAGT
`FLD0331 AGGATTGAGG
`FLD0332 CCAGAACAGA
`FLD0333 CGTCAAGCAT
`FLD0334 TTGTCGAGAC
`FLD0335 GACAGGTGAC
`FLD0336 CTGACAAGTG
`FLD0337 CACGAAGAGC
`FLD0338 CATACCTGAT
`FLD0339 GACGTGCTTC
`FLD0340 ATTGTGGAGT
`FLD0341 TCTGGTCTCA
`FLD0342 AGGTAAGAGG
`FLD0343 TCCTGACAGA
`FLD0344 GCACTGTTGC
`FLD0345 ACCATGAGTC
`FLD0346 AATGCAGTGT
`FLD0347 ATATGGTGGA
`FLD0348 ACTCAGTTAC
`FLD0349 AAGTGCGATG
`FLD0350 CCACAGAGTG
`FLD0351 AGTGGTGATC
`FLD0352 ACTTCTTAGC
`FLD0353 GCCACATATA
`FLD0354 ACGCAGGAGT
`FLD0355 AATATGCTGC
`FLD0356 AAGCGTAGAA
`FLD0357 GACAGCAAGC
`FLD0358 CTGACCGAGA
`FLD0359 CGCGACTTGT
`FLD0360 CATCAACATG
`FLD0361 TGGCTACGCT
`FLD0362 ACGCGGACTA
`FLD0363 AGAGGTCGGA
`FLD0364 AATCGAGCGT
`FLD0365 AAGTACACTC
`FLD0366 AGCTGAATGA
`FLD0367 ATGCCTATCA
`FLD0368 ACTGTAGGAC
`FLD0369 ATAGCCGTGT
`FLD0370 TCACGACGAA
`FLD0371 ATCTGTCCAT
`FLD0372 ACTTAGAGAG
`FLD0373 AGTGGCAGGT
`FLD0374 ATGAGGTCGT
`FLD0375 AGGAGAAGGA
`FLD0376 ACAACTGCAA
`FLD0377 ATTAGCGAGT
`FLD0378 ACAACGAACA
`FLD0379 AGAGCGCCAA
`FLD0380 AGGTAGCTCA
`FLD0381 AACGCCAAGA
`FLD0382 AAGGTATGAG
`FLD0383 ATGGAGCACT
`FLD0384 ACGGTGCTAG
`
`
`
`12
`
`Foresight EX1032
`Foresight v Personalis
`
`

`

`Table S3: Mutations identified in FFPE samples. Diagnosis: type (FT, fallopian tube; O, ovarian; PP, primary peritoneal), histiotype (END,
`endometrioid, grade III; HGSOC, high-grade serous ovarian carcinoma; S/CC, mixed serous and clear cell; S/END, mixed serous papillary and
`endometrioid, high grade), and stage. FFPE source: sample type (B, biopsy; IDS, interval-debulking surgery; PS, primary surgery), sample tiss