Partial UDG-treatment for screening of ancient DNA

Transcription

1 Electronic Supplementary Material Partial UDG-treatment for screening of ancient DNA Nadin Rohland 1,2,*, Eadaoin Harney 1,2,3, Swapan Mallick 1,2,3, Susanne Nordenfelt 1,2 and David Reich 1,2,3 1 Department of Genetics, Harvard Medical School, Boston, MA Broad Institute of MIT and Harvard, Cambridge, MA Howard Hughes Medical Institute, Boston, MA * To whom correspondence should be addressed nrohland@genetics.med.harvard.edu

2 Library preparation with partial UDG treatment Reagents and consumables Water Sigma W4502 Tween-20 Sigma P ML 5M NaCl Sigma S5150-1L dntp Mix (25mM each) ThermoFisher FERR1121 ATP (100mM) ThermoFisher FERR x Buffer Tango ThermoFisher FERRBY5 T4 Polynucleotide Kinase (10 U/uL) ThermoFisher FEREK0032 T4 DNA Polymerase (5 U/uL) ThermoFisher FEREP0062 T4 DNA Ligase (5 U/uL) ThermoFisher FEREL0011 USER enzyme NEB M5505L UGI (Uracil Glycosylase Inhibitor) NEB M0281L Bst DNA Polymerase, large frag. NEB M0275S ThermoPol reaction buffer NEB B9004S MinElute PCR Purification Kit Qiagen Collection tubes Qiagen mL LoBind tubes VWR mL LoBind tubes VWR Tube Strip of 0.2 ml Tubes VWR Pfu Turbo Cx Hotstart DNA Polymerase Agilent Technologies M Tris-HCl ph 8.0 ThermoFisher AM M EDTA ph 8.0 (BioExpress) VWR well plates ThermoFisher Cap Strips ThermoFisher. N PreHyb-F 5 -CTTTCCCTACACGACGCTCTTC-3 PreHyb-R 5 -GTGACTGGAGTTCAGACGTGTGCT -3 Barcoded P5 adapter (see Table S3) Barcoded P7 adapter (see Table S3)!!!! 10 mm Tris-HCl volume final concentration H 2 O 49.5 ml 1M Tris-HCl, ph ml 10mM UV-irradiate for 30min using a UV-cross-linker (254nm). TE buffer volume final concentration H 2 O 49.4 ml 1M Tris-HCl, ph ml 10 mm 0.5M EDTA, ph ml 1 mm UV-irradiate for 30min using a UV-cross-linker (254nm).!! TTE buffer volume final concentration TE Buffer ml 1x Tween µl 0.05% UV-irradiate for 30min using a UV-cross-linker (254nm). 2

3 Adapter hybridization Needs to be done initially or when adapter-cross-contamination is observed. Adapters will last for ~500 preparations per barcode. 1) Assemble the following hybridization reactions (one well per barcoded adapter, keep P5 and P7 adapters strictly separate) and use plastic caps to close. volume in µl final concentration per adapter in 50 µl Hybridization mix for P5 adapter (100 µm) 1-P5.F (500 µm) µm 1-P5/P7.R (500 µm) µm TE x 5M NaCl mM volume in µl final concentration per adapter in 50 µl Hybridization mix for P7 adapter (100 µm) 1-P7.F (500 µm) µm 1-P5/P7.R (500 µm) µm TE x 5M NaCl mM 2) Mix, centrifuge and incubate in a thermal cycler with heated lid for 10 sec at 95 C, followed by a ramp from 95 C to 12 C at a rate of 0.1 C/s. 3) Take an aliquot and dilute 10-fold with TE to obtain individual 10µM solutions of P5- and P7-adapters these are the ready-to-use adapters and can be used for many reactions per barcode (1 µl per ligation). To reduce risk of cross-contamination, consider using 8-strip tubes with individual caps or 0.5mL tubes for readyto-use adapters so each adapter can be opened independently. 3

4 Library construction with partial UDG treatment - protocol Include at least one library negative control per batch and it is recommended that a positive control with the same barcode combination is used each time a batch of libraries is constructed; two complementary oligonucleotides (hybridized according to the same procedure used for the adapters) may serve best for this. Partial USER treatment 1) Prepare a reaction mix as shown below (use 0.5 ml tubes). volume in µl final concentration per reaction per 60 µl reaction Buffer Tango (10x) 6 1 x dntp s (25 mm each) µm each ATP (100 mm) mm USER (1 U/µL) U/µL H 2 O DNA extract total 52.2 Mix carefully by flicking the tube with a finger after extract is added and spin quickly. Incubate in a thermal cycler for 30min at 37 C. UDG inhibition 2) Add to each reaction: UGI (2U/µL) U/ µl total 55.8 After adding UGI, mix by flicking the tube with a finger, spin, and incubate in thermal cycler for 30min at 37 C, after a quick spin. Blunt end repair 3) Add to each reaction: T4 PNK (10 U/µL) U/µL T4 Polymerase (5U/µL) U/µL total 60 After adding both enzymes, mix by flicking tube with a finger, spin, and incubate for 15 min at 25 C, followed by 5 min at 12 C. Reaction cleanup using the MinElute PCR purification Kit 4) Add five volumes of buffer PB to the reaction and mix (for a 60 µl reaction add 300 µl of PB). Transfer mix to a MinElute column placed in a collection tube. Centrifuge for 30 sec at 3,300 x g. Discard flowthrough and collection tube. Add 700 µl PE buffer to the column. Centrifuge for 30 sec at max. speed. Discard flowthrough. Repeat this wash step. Dry spin for 1 min at max. speed. Place MinElute column into new 1.5 ml tube. Add 18 µl 10mM Tris-HCl to the silica membrane. Let sit for 5 min and then centrifuge for 1 min at max. speed. Discard MinElute column. 4

5 Adapter ligation 5) Chose a unique barcode combination for each sample; do not use any adapter multiple times per batch. Add 1 µl of respective P5- AND P7-adapter (hybridized) to 1.5 ml tube from 4). Properly mix DNA and adapter before adding the remaining components. PEG is highly viscous, vortex the master mix before adding T4 DNA ligase and mix again thereafter by flicking the tube with a finger. volume in µl finalconcentration per reaction per reaction H 2 O 12 T4 DNA ligase buffer (10x) 4 1x PEG-4000 (50%) 4 5% P5-adapter (10 µm) (1) 0.25 µm P7-adapter (10 µm) (1) 0.25 µm Blunted DNA (step 4) (17) T4 DNA ligase (5 U/ µl) U/µL Total 40 Mix by flicking the tube with a finger, spin and incubate for 30 min at room temperature. Reaction cleanup using the MinElute PCR purification Kit 6) Perform purification of DNA with MinElute columns exactly as described in step 2, but elute in 20 µl 10 mm Tris-HCl. Adapter fill-in 7) Prepare a reaction mix as shown below (you have to transfer the ligation product from the 1.5mL tube into a tube that fits the thermal cycler). volume in µl final concentration per reaction per reaction H 2 O 13.6 ThermoPol buffer (10x) 4 1x dntp s (25mM each) µm each Bst Polymerase, large fragment (8 U/µL) U/µL adapter-ligated product (step 6) (20) Total 40 Mix by flicking the tube with a finger, spin, and incubate for 20 min at 37 C, followed by 20 min at 80 C. Optional The approximate number of unique molecules in each library can be estimated by performing a quantitative PCR of a proportion of the fill-in product with primers annealing to the universal adapter ends. Amplification 8) Prepare below PCR mix for each library using universal PreHyb-primers and Pfu Turbo Cx Hotstart DNA Polymerase, include some PCR negatives. 5

6 volume in µl per sample final concentration per reaction H 2 O Pfu Turbo Cx React. Buf. (10x) 40 1x PreHyb-F (10 µm) µm PreHyb-R (10µM) µm dntp s (25mM each) mM each Pfu Turbo Cx Hotstart DNA Polymerase (2.5 U/µL) 8 5 U Library (step 7) (39) Total 400 Distribute 8 x 50 µl (or 4 x 100 µl ) PCR mix into PCR plate, seal with PCR cover and leave the clean-room. Initial denaturation for 2min at 95 C, followed by 30 cycles with 30sec at 95 C, 30sec at 55 C and 1min at 72 C, final extension at 72 C for 10min. Reaction cleanup using the MinElute PCR purification Kit 9) Add 2 ml PB to collapsed 400 µl PCR reaction and split over 2 MinElute columns, proceed as usual (2x PE-wash, dry spin at max. speed) and elute in 25 µl TTE each, pool both eluats per library = 50 µl. 10) Perform Nanodrop measurement for concentration assessment and BioAnalyzer or aragose gel to see if preparation worked in general, to verify the size distribution typical for ancient DNA libraries and that no major adapter artifacts are present. 6

7 Table S1: Oligonucleotides used in this study. Adapters, primer and blocking oligonucleotides. ID barcode P5.F (5' 3') P7.F (5' 3') P5/P7.R (5' 3') 1 ATCGATT CTTTCCCTACACGACGCTCTTCCGATCTatcgatt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTatcgatt aatcgatagatcg 2 CAGTCAA CTTTCCCTACACGACGCTCTTCCGATCTcagtcaa GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcagtcaa ttgactgagatcg 3 GCTAGCC CTTTCCCTACACGACGCTCTTCCGATCTgctagcc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgctagcc ggctagcagatcg 4 TGACTGG CTTTCCCTACACGACGCTCTTCCGATCTtgactgg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtgactgg ccagtcaagatcg 5 CAATTGC CTTTCCCTACACGACGCTCTTCCGATCTcaattgc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcaattgc gcaattgagatcg 6 GCCAATG CTTTCCCTACACGACGCTCTTCCGATCTgccaatg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgccaatg cattggcagatcg 7 TGGCCAT CTTTCCCTACACGACGCTCTTCCGATCTtggccat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtggccat atggccaagatcg 8 ATTGGCA CTTTCCCTACACGACGCTCTTCCGATCTattggca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTattggca tgccaatagatcg 9 CGATGTA CTTTCCCTACACGACGCTCTTCCGATCTcgatgta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgatgta tacatcgagatcg 10 GTCATAC CTTTCCCTACACGACGCTCTTCCGATCTgtcatac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtcatac gtatgacagatcg 11 TAGCACG CTTTCCCTACACGACGCTCTTCCGATCTtagcacg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtagcacg cgtgctaagatcg 12 ACTGCGT CTTTCCCTACACGACGCTCTTCCGATCTactgcgt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTactgcgt acgcagtagatcg 13 GGTATCG CTTTCCCTACACGACGCTCTTCCGATCTggtatcg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTggtatcg cgataccagatcg 14 TTACAGT CTTTCCCTACACGACGCTCTTCCGATCTttacagt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTttacagt actgtaaagatcg 15 AACGCTA CTTTCCCTACACGACGCTCTTCCGATCTaacgcta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaacgcta tagcgttagatcg 16 CCGTGAC CTTTCCCTACACGACGCTCTTCCGATCTccgtgac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTccgtgac gtcacggagatcg 17 CGCTGAG CTTTCCCTACACGACGCTCTTCCGATCTcgctgag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgctgag ctcagcgagatcg 18 GTGATCT CTTTCCCTACACGACGCTCTTCCGATCTgtgatct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtgatct agatcacagatcg 19 TATCAGA CTTTCCCTACACGACGCTCTTCCGATCTtatcaga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtatcaga tctgataagatcg 20 ACAGCTC CTTTCCCTACACGACGCTCTTCCGATCTacagctc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTacagctc gagctgtagatcg 21 ACGGTCT CTTTCCCTACACGACGCTCTTCCGATCTacggtct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTacggtct agaccgtagatcg 22 CGTTAGA CTTTCCCTACACGACGCTCTTCCGATCTcgttaga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgttaga tctaacgagatcg 23 GTAACTC CTTTCCCTACACGACGCTCTTCCGATCTgtaactc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtaactc gagttacagatcg 24 TACCGAG CTTTCCCTACACGACGCTCTTCCGATCTtaccgag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtaccgag ctcggtaagatcg 25 TACGTTC CTTTCCCTACACGACGCTCTTCCGATCTtacgttc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtacgttc gaacgtaagatcg 26 ACGTAAG CTTTCCCTACACGACGCTCTTCCGATCTacgtaag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTacgtaag cttacgtagatcg 27 CGTACCT CTTTCCCTACACGACGCTCTTCCGATCTcgtacct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgtacct aggtacgagatcg 28 GTACGGA CTTTCCCTACACGACGCTCTTCCGATCTgtacgga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtacgga tccgtacagatcg 29 CTACTCG CTTTCCCTACACGACGCTCTTCCGATCTctactcg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctactcg cgagtagagatcg 30 GACGAGT CTTTCCCTACACGACGCTCTTCCGATCTgacgagt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgacgagt actcgtcagatcg 31 TCGTCTA CTTTCCCTACACGACGCTCTTCCGATCTtcgtcta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcgtcta tagacgaagatcg 32 AGTAGAC CTTTCCCTACACGACGCTCTTCCGATCTagtagac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagtagac gtctactagatcg 33 GGAGTAC CTTTCCCTACACGACGCTCTTCCGATCTggagtac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTggagtac gtactccagatcg 34 TTCTACG CTTTCCCTACACGACGCTCTTCCGATCTttctacg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTttctacg cgtagaaagatcg 35 AAGACGT CTTTCCCTACACGACGCTCTTCCGATCTaagacgt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaagacgt acgtcttagatcg 36 CCTCGTA CTTTCCCTACACGACGCTCTTCCGATCTcctcgta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcctcgta tacgaggagatcg 37 ACTTGGC CTTTCCCTACACGACGCTCTTCCGATCTacttggc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTacttggc gccaagtagatcg 38 CGAATTG CTTTCCCTACACGACGCTCTTCCGATCTcgaattg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgaattg caattcgagatcg 39 GTCCAAT CTTTCCCTACACGACGCTCTTCCGATCTgtccaat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtccaat attggacagatcg 40 TAGGCCA CTTTCCCTACACGACGCTCTTCCGATCTtaggcca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtaggcca tggcctaagatcg 41 AGATTCC CTTTCCCTACACGACGCTCTTCCGATCTagattcc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagattcc ggaatctagatcg 42 CTCAAGG CTTTCCCTACACGACGCTCTTCCGATCTctcaagg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctcaagg ccttgagagatcg 43 GAGCCTT CTTTCCCTACACGACGCTCTTCCGATCTgagcctt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgagcctt aaggctcagatcg 44 TCTGGAA CTTTCCCTACACGACGCTCTTCCGATCTtctggaa GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtctggaa ttccagaagatcg 45 CTAGACA CTTTCCCTACACGACGCTCTTCCGATCTctagaca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctagaca tgtctagagatcg 46 GACTCGC CTTTCCCTACACGACGCTCTTCCGATCTgactcgc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgactcgc gcgagtcagatcg 47 TCGAGTG CTTTCCCTACACGACGCTCTTCCGATCTtcgagtg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcgagtg cactcgaagatcg 48 AGTCTAT CTTTCCCTACACGACGCTCTTCCGATCTagtctat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagtctat atagactagatcg 49 CCTGCAG CTTTCCCTACACGACGCTCTTCCGATCTcctgcag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcctgcag ctgcaggagatcg 50 GGATGCT CTTTCCCTACACGACGCTCTTCCGATCTggatgct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTggatgct agcatccagatcg 51 TTCATGA CTTTCCCTACACGACGCTCTTCCGATCTttcatga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTttcatga tcatgaaagatcg 52 AAGCATC CTTTCCCTACACGACGCTCTTCCGATCTaagcatc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcatc gatgcttagatcg 53 CTAACAG CTTTCCCTACACGACGCTCTTCCGATCTctaacag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctaacag ctgttagagatcg 54 GACCGCT CTTTCCCTACACGACGCTCTTCCGATCTgaccgct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgaccgct agcggtcagatcg 55 TCGGTGA CTTTCCCTACACGACGCTCTTCCGATCTtcggtga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcggtga tcaccgaagatcg 56 AGTTATC CTTTCCCTACACGACGCTCTTCCGATCTagttatc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagttatc gataactagatcg 57 AGTCACG CTTTCCCTACACGACGCTCTTCCGATCTagtcacg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagtcacg cgtgactagatcg 58 CTAGCGT CTTTCCCTACACGACGCTCTTCCGATCTctagcgt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctagcgt acgctagagatcg 59 GACTGTA CTTTCCCTACACGACGCTCTTCCGATCTgactgta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgactgta tacagtcagatcg 60 TCGATAC CTTTCCCTACACGACGCTCTTCCGATCTtcgatac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcgatac gtatcgaagatcg 7

8 ID barcode P5.F (5' 3') P7.F (5' 3') P5/P7.R (5' 3') 61 CGCCATG CTTTCCCTACACGACGCTCTTCCGATCTcgccatg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcgccatg catggcgagatcg 62 GTGGCAT CTTTCCCTACACGACGCTCTTCCGATCTgtggcat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgtggcat atgccacagatcg 63 TATTGCA CTTTCCCTACACGACGCTCTTCCGATCTtattgca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtattgca tgcaataagatcg 64 ACAATGC CTTTCCCTACACGACGCTCTTCCGATCTacaatgc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTacaatgc gcattgtagatcg 65 AGGCCTA CTTTCCCTACACGACGCTCTTCCGATCTaggccta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaggccta taggcctagatcg 66 CTTGGAC CTTTCCCTACACGACGCTCTTCCGATCTcttggac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcttggac gtccaagagatcg 67 GAATTCG CTTTCCCTACACGACGCTCTTCCGATCTgaattcg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgaattcg cgaattcagatcg 68 TCCAAGT CTTTCCCTACACGACGCTCTTCCGATCTtccaagt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtccaagt acttggaagatcg 69 CCTTACG CTTTCCCTACACGACGCTCTTCCGATCTccttacg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTccttacg cgtaaggagatcg 70 GGAACGT CTTTCCCTACACGACGCTCTTCCGATCTggaacgt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTggaacgt acgttccagatcg 71 TTCCGTA CTTTCCCTACACGACGCTCTTCCGATCTttccgta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTttccgta tacggaaagatcg 72 AAGGTAC CTTTCCCTACACGACGCTCTTCCGATCTaaggtac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaaggtac gtaccttagatcg 73 CATAGGC CTTTCCCTACACGACGCTCTTCCGATCTcataggc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcataggc gcctatgagatcg 74 GCACTTG CTTTCCCTACACGACGCTCTTCCGATCTgcacttg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcacttg caagtgcagatcg 75 TGCGAAT CTTTCCCTACACGACGCTCTTCCGATCTtgcgaat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtgcgaat attcgcaagatcg 76 ATGTCCA CTTTCCCTACACGACGCTCTTCCGATCTatgtcca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTatgtcca tggacatagatcg 77 GTTGACT CTTTCCCTACACGACGCTCTTCCGATCTgttgact GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgttgact agtcaacagatcg 78 TAATCGA CTTTCCCTACACGACGCTCTTCCGATCTtaatcga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtaatcga tcgattaagatcg 79 ACCAGTC CTTTCCCTACACGACGCTCTTCCGATCTaccagtc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaccagtc gactggtagatcg 80 CGGCTAG CTTTCCCTACACGACGCTCTTCCGATCTcggctag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcggctag ctagccgagatcg 81 ATACGAC CTTTCCCTACACGACGCTCTTCCGATCTatacgac GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTatacgac gtcgtatagatcg 82 CACGTCG CTTTCCCTACACGACGCTCTTCCGATCTcacgtcg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcacgtcg cgacgtgagatcg 83 GCGTAGT CTTTCCCTACACGACGCTCTTCCGATCTgcgtagt GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcgtagt actacgcagatcg 84 TGTACTA CTTTCCCTACACGACGCTCTTCCGATCTtgtacta GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtgtacta tagtacaagatcg 85 CTCTGGA CTTTCCCTACACGACGCTCTTCCGATCTctctgga GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctctgga tccagagagatcg 86 GAGATTC CTTTCCCTACACGACGCTCTTCCGATCTgagattc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgagattc gaatctcagatcg 87 TCTCAAG CTTTCCCTACACGACGCTCTTCCGATCTtctcaag GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtctcaag cttgagaagatcg 88 AGAGCCT CTTTCCCTACACGACGCTCTTCCGATCTagagcct GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagagcct aggctctagatcg 89 AGCATCA CTTTCCCTACACGACGCTCTTCCGATCTagcatca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagcatca tgatgctagatcg 90 CTGCAGC CTTTCCCTACACGACGCTCTTCCGATCTctgcagc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgcagc gctgcagagatcg 91 GATGCTG CTTTCCCTACACGACGCTCTTCCGATCTgatgctg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgatgctg cagcatcagatcg 92 TCATGAT CTTTCCCTACACGACGCTCTTCCGATCTtcatgat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcatgat atcatgaagatcg 93 CAGGAAT CTTTCCCTACACGACGCTCTTCCGATCTcaggaat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcaggaat attcctgagatcg 94 GCTTCCA CTTTCCCTACACGACGCTCTTCCGATCTgcttcca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcttcca tggaagcagatcg 95 TGAAGGC CTTTCCCTACACGACGCTCTTCCGATCTtgaaggc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtgaaggc gccttcaagatcg 96 ATCCTTG CTTTCCCTACACGACGCTCTTCCGATCTatccttg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTatccttg caaggatagatcg 97 GACTTAT CTTTCCCTACACGACGCTCTTCCGATCTgacttat GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgacttat ataagtcagatcg 98 TCGAACA CTTTCCCTACACGACGCTCTTCCGATCTtcgaaca GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtcgaaca tgttcgaagatcg 99 AGTCCGC CTTTCCCTACACGACGCTCTTCCGATCTagtccgc GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTagtccgc gcggactagatcg 100 CTAGGTG CTTTCCCTACACGACGCTCTTCCGATCTctaggtg GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTctaggtg cacctagagatcg UNIVERSAL ADAPTER IS1_adapter.P5 A*C*A*C*TCTTTCCCTACACGACGCTCTTCCG*A*T*C*T IS2_adapter.P7 G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCG*A*T*C*T IS3_adapter.P5+P7 A*G*A*T*CGGAA*G*A*G*C * PTO bonds PRIMER 5' 3' annealing temperature in C capture setup PreHyb-F CTTTCCCTACACGACGCTCTTC 55 short, intermediate PreHyb-R GTGACTGGAGTTCAGACGTGTGCT 55 short indexing P5 AATGATACGGCGACCACCGAGATCTACACxxxxxxxACACTCTTTCCCTACACGACGCTC 62 long indexing P7 CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGT 62 intermediate, long IS5_reamp.P5 AATGATACGGCGACCACCGA 60 long IS6_reamp.P7 CAAGCAGAAGACGGCATACGA 60 intermediate, long BLOCKING OLIGOS 5' 3' capture setup Block_short_P5.R AGATCGGAAGAGCGTCGTGTAGGGAAAG short, intermediate Block_short_P7_Multi.F AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC short, intermediate, long Block_P7.F.part2 ATCTCGTATGCCGTCTTCTGCTTG intermediate, long Block_P5.R.part1 GTGTAGATCTCGGTGGTCGCCGTATCATT long Block_P5.R.part2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT long 8

9 Table S2: All possible substitution rates in the terminal 25 nucleotides of 2 samples (a and b) for three different library preparations. UDG- (no UDG treatment - I), pudg (partial UDG treatment - II) and UDG+ (full UDG treatment - III). a position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T UDG I a position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T pudg II a position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T UDG III

10 b position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T UDG I b position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T pudg II

11 b position G>A C>T A>G T>C A>C A>T C>G C>A T>G T>A G>C G>T UDG III

12 Table S3: Basic statistics of the 60 libraries (a to bh) and the negative controls, both before and after enrichment for. before enrichment after enrichment sample ID indirect age in ybc direct age in calbc in demultiplexed fastq file with matching barcodes with correct barcode combination and merged R1 and R2 to to in demultiplexed fastq file with matching barcodes with correct barcode combination and merged R1 and R2 12 alignin g to mtdn A to unique alignin g to unique to damage rate in terminal bases coverage on contaminati on estimation - fraction authentic a ,702 99% 10, % 108,608 98% 105,304 61, % 16, % 8.83% [ 0.994,1.0 ] b ,924 99% 10, % 111,334 99% 109,007 81, % 22, % 9.30% [ 0.985,0.993 ] c , % 16, % 153,915 99% 151,513 86, % 4, % 6.49% [ 0.911,0.955 ] d ,420 62% 5, % 128,896 86% 98,059 68, % 9, % 13.26% [ 0.993,0.998 ] e ,115 99% 16, % 168,509 98% 156,128 5, % 2, % 10.52% [ 0.982,1.0 ] f ,565 99% 15, % 198,643 98% 181, , % 22, % 11.46% [ 0.993,0.997 ] g ,128 99% 12, % 192,164 98% 169, , % 15, % 9.56% [ 0.973,0.982 ] h , % 6, % 202,443 99% 193, , % 9, % 8.26% [ 0.956,0.967 ] i ,625 98% 11, % 174,565 94% 142,091 94, % 17, % 10.40% [ 0.989,0.995 ] j ,598 99% 12, % 159,062 98% 136,838 82, % 13, % 6.00% [ 0.960,0.974 ] k ,537 99% 17, % 189,203 98% 172,723 94, % 6, % 11.01% [ 0.990,0.995 ] l ,251 99% 9, % 169,148 99% 151,824 82, % 4, % 6.61% [ 0.983,0.989 ] m ,545 99% 9, % 192,147 99% 173, , % 25, % 10.27% [ 0.995,0.997 ] n ,571 99% 13, % 172,725 99% 155, , % 11, % 5.35% [ 0.980,0.986 ] o ,676 99% 12, % 206,170 99% 183, , % 26, % 5.98% [ 0.974,0.981 ] p ,682 99% 14, % 163,530 98% 151, , % 26, % 6.13% [ 0.997,1.0 ] q ,689 99% 9, % 174,733 98% 165, , % 21, % 15.21% [ 0.998,1.0 ] r ,523 99% 14, % 188,856 99% 172, , % 20, % 5.63% [ 0.994,0.998 ] s , % 18, % 191,366 98% 179, , % 16, % 9.40% [ 0.997,1.0 ] t ,443 99% 13, % 193,703 97% 166,994 32, % 10, % 9.21% [ 0.985,0.994 ] u ,337 99% 36, % 545,753 99% 487, , % 31, % 7.99% NA NA v ,631 99% 37, % 473,526 99% 421, , % 31, % 12.35% NA NA w ,691 99% 27, % 417,853 99% 362, , % 28, % 6.30% NA NA x ,101 99% 16, % 228,285 98% 209, , % 26, % 5.10% NA NA y ,498 99% 12, % 187,905 99% 168, , % 22, % 8.77% [ 0.985,0.991 ] z ,168 99% 10, % 182,978 96% 163, , % 21, % 7.65% [ 0.998,1.0 ] aa ,071 98% 8, % 203,348 95% 181, , % 13, % 9.50% [ 0.984,0.989 ] ab ,000 98% 15, % 192,131 97% 175, , % 7, % 9.99% [ 0.995,1.0 ] ac ,046 99% 19, % 220,069 98% 203, , % 4, % 9.64% [ 0.986,0.991 ] ad ,158 99% 12, % 192,331 97% 172, , % 26, % 6.79% [ 0.997,1.0 ] ae ,771 99% 7, % 147,797 97% 132, , % 24, % 7.40% [ 0.997,0.999 ] af ,968 99% 12, % 144,319 97% 128,219 96, % 23, % 11.14% [ 0.990,0.995 ] ag ,609 99% 8, % 126,307 97% 111,205 78, % 17, % 4.38% [ 0.993,0.998 ] ah ,499 99% 9, % 140,659 96% 130,583 77, % 8, % 8.27% [ 0.999,1.0 ] ai , % 15, % 181,771 99% 163, , % 27, % 6.02% [ 0.991,0.996 ] aj ,727 99% 17, % 215,002 97% 198, , % 25, % 10.36% [ 0.998,1.0 ] ak ,558 99% 14, % 201,058 98% 185, , % 21, % 4.41% [ 0.993,0.998 ] CI

13 al ,876 98% 11, % 154,831 96% 135,511 99, % 17, % 8.03% [ 0.992,0.996 ] am ,135 99% 15, % 227,220 97% 207, , % 8, % 5.66% [ 0.999,1.0 ] an ,048 99% 52, % 503,279 99% 460, , % 30, % 6.09% NA NA ao ,205 98% 33, % 514,214 98% 456, , % 31, % 7.78% NA NA ap ,206 99% 14, % 223,311 98% 206, , % 28, % 8.27% NA NA aq ,459 99% 11, % 222,745 98% 204, , % 28, % 10.95% NA NA ar ,286 98% 15, % 178,726 97% 159, , % 24, % 11.45% [ 0.997,0.999 ] as ,885 99% 16, % 220,655 99% 207, , % 24, % 7.57% [ 0.998,1.0 ] at ,929 99% 13, % 227,876 97% 213, , % 24, % 11.92% 92.2 NA NA au ,844 99% 10, % 170,078 98% 154, , % 22, % 8.94% [ 0.990,0.994 ] av ,817 99% 21, % 231,651 98% 208, , % 4, % 7.77% [ 0.941,0.954 ] aw ,656 99% 9, % 190,247 98% 174, , % 1, % 6.87% 4.5 ax ,115 99% 20, % 199,274 98% 182, , % 1, % 4.74% 4.1 ay NA 9,244 99% 9, % 227,332 98% 206, , % 1, % 4.40% 3.9 az ,119 97% 12, % 114,098 95% 107,026 66, % % 3.47% 1.0 ba ,337 97% 12, % 198,914 93% 168, , % % 9.36% 0.9 bb ,629 99% 15, % 206,815 98% 196, , % % 8.98% 0.4 bc ,347 86% 7, % 238,715 90% 209,102 86, % % 15.26% 0.1 bd ,071 98% 13, % 162,123 98% 145,022 39, % % 0.00% 0.1 be ,919 99% 9, % 162,689 98% 143,516 45, % % 5.56% 0.1 bf ,986 95% 13, % 162,082 97% 151,720 44, % % 0.00% 0.0 bg ,666 97% 15, % 175,192 93% 151,232 42, % % 0.00% 0.0 bh ,622 99% 16, % 157,313 97% 140,567 1, % % 0.00% 0.0 batch ID control 1 Extraction Negative in demultiplexed fastq file with matching barcodes before enrichment with correct barcode combination and merged R1 and R2 to hg19 Reads to in demultiplexed fastq file with matching barcodes with correct barcode combination and merged R1 and R2 13 after enrichment to to unique to unique to 6% % 13,668 7,060 52% % Library Negative 8% % 2,452 2,205 90% 4 0.2% 0 2 Extraction Negative 8% % 24,640 11,718 48% % 0 2 Library Negative 5% % 2, % 2 0.1% 0 3 Extraction Negative 6% % 8,766 4,147 47% % 0 3 Library Negative 5% % 3,645 3,320 91% 2 0.1% 0 4 Extraction Negative 4% % 35,226 21,763 62% % 0 4 Library Negative 4% % % 4 1.6% 0 13, ,991 5 Extraction Negative 8% % 10,014 5,276 53% % Library Negative 5% % 2, % 1 0.0% 0 6 Extraction Negative 11% 1, % 29,783 12,165 41% % 0 6 Library Negative 6% % 1, % 4 0.2% 0 7 Extraction Negative 9% 1, % 19,536 5,236 27% % 0 7 Library Negative 0% % 2,908 1,710 59% % 0 8 Extraction Negative 6% % 16,312 7,907 48% % Library Negative 1% % 2,128 1,813 85% % 0 9 Extraction Negative 64% 4, % 292, ,403 43% % Library Negative 23% % 65,358 47,099 72% 6 0.0% , , Extraction Negative 9% % 74,426 37,788 51% % Library Negative 0% % 1,789 1,737 97% 4 0.2% 0 11 Extraction Negative 6,684 35% ,962 81% 88,529 49,312 56% % 0.01 damage rate in terminal bases

14 I II III Figure S1. Fragmentation pattern plots from MapDamage2.0 of sample a after three library preparations (I no UDG treatment, II partial UDG treatment, III full UDG treatment) and enrichment. Fragmentation plots of partial UDGtreated libraries are similar to full UDG-treated libraries with elevated C s at the position in the reference before the fragment and elevated G s at the position in the reference after the fragment. 14