DNA Samples and Sanger Sequencing

Supplementary files:

DNA samples and Sanger sequencing.

The Hospital Universitario Central Asturias (HUCA) is a reference center for the genetic studies of hereditary cardiac diseases in Spain. As part of the routine work patients diagnosed with LQTS/BS are sequenced for the KCNH2, KCNQ1, KCNE1, KCNE2 and SCN5A. Briefly, DNA was obtained following a salting-out method, resuspended in water to a final concentration of 500 ng/µl, and frozen till use. The coding exons plus at least 10 nucleotides of the flanking intronic bases were PCR amplified with primers that matched intron sequences. All the exons were amplified in single 300-700 bp fragments (except exon 26 of SCN5A that was amplified in three overlapping fragments), and PCR fragments were purified and the two strands sequenced with BigDye chemistry in an ABI3130xl equipment. The full detailed procedure (including primer sequences) is available upon request to the corresponding author.

DNA template preparation.

Genomic DNA thawed and an aliquot was adjusted to a concentration of 10 ng/µl using Real Time Taqman quantification with RNase P Detection Reagents (FAM™) (Life technologies) in a 7500 Reat Time PCR-System (Applied Biosystems). A pool 10µl of each of the 20 assayed DNAs was created and stored at 4ºC for two days prior to the Ampliseq amplification.

Multiplex Ampliseq amplification.

Two amplifications were performed with the Ion Ampliseq™ Library Kit in conjunction with Ion Ampliseq™ Custom Primer Pool protocols, according to the manufacturer procedures (Life Technologies) following the next sequential steps: polymerase chain reaction (PCR) of the pooled DNAs in two Ampliseq tubes, partial digestion of the primer sequence with FuPa Reagent, ligation of the adapters, purification by Agencourt® AMPure® XP Reagent, PCR with the adapters using Platinum® PCR SuperMix High Fidelity enzyme (Invitrogen), purification by Agencourt® AMPure® XP Reagent, quantification of the sample (Agilent Bioanalyser Instrument and Qubit® 2.0 Fluorometer), and dilution of the sample to a final concentration of 20 pM.

Amplicons sequencing.

Template preparation, emulsion PCR, emulsion breaking, and enrichment was performed using the Ion PGM™ Template OT2 200 kit following manufacturer procedures (Life Technologies). This consisted on the next sequential steps: emPCR in the One Touch Instrument, purification by magnet resins in the Ion Touch ES, and quality control using the Ion Sphere™ Quality Control Assay. All amplicons were sequenced using the Ion Torrent PGM™ sequencer.

Data analysis.

Data were processed using the Ion Torrent platform-specific pipeline software Torrent Suite v3.4.2 (Life technologies), to generate sequences reads filtered according by pipeline software quality controls. Reads assembling and variant identification were performed with the Variant Caller v3.4.51874 software (Life Technologies) using FastQ files containing sequence reads and the Ion Ampliseq Designer BED file software to map the amplicons. Integrative Genome Viewer (IGV, Broad Institute) was used for the analysis of depth coverage, sequence quality, and variants identification. The somatic sample Variant Caller default algorithm was use for variant identification.

Suppl. table 1. Pooled primers of the Ampliseq.

Pool / Fordward Primer (5’-3’) / Reverse Primer (5’-3’) / Gene / Amplicon Start / Amplicon Stop
A / GTTGGGTTGTTCTATGGCCAGAT / CTCATGGTACTGGGATTCTTCGG / KCNE1 / 35821576 / 35821791
A / CGCTGTGGTGTTAGACAGGAT / GGTTTTGGCCAATACGTACTTTTTGATT / KCNE1 / 35821909 / 35822033
A / CGTTTTCCTAACCTTGTTCGCCTATT / CCATGAGGTACAGGATGACATAGTAGA / KCNE2 / 35742722 / 35742940
A / GCAGGAAAAGTACAAGAGCCAAATC / GCAAGGAACTCACTTTCTTCTAAGCAA / KCNE2 / 35743053 / 35743257
A / TGGTTGGCCAATAAAGATAGATGACAT / GTAAATCTCTCCTTTCTATCCCCACAG / KCNE2 / 35743288 / 35743486
A / GAGCCACGTGTCCACACT / CCTCTTCGCCTGCCCAT / KCNH2 / 150642424 / 150642637
A / CCACCTGCACTCCCTCA / CTGCAGGCGCCTTCTCA / KCNH2 / 150644398 / 150644608
A / TGCAAGTGTCGCTGCTCTT / GGAGAGCCCGTCCAGTG / KCNH2 / 150644706 / 150644875
A / GAGGCCTGGGTAAAGCAGA / AAGGGCCCTGATACTGATTTTGG / KCNH2 / 150645437 / 150645660
A / GACCAGAAGTGGTCGGAGAA / AGCTCCCTGTCCTCTCCAT / KCNH2 / 150645975 / 150646165
A / GTCTGTTTGTGGCGGATCCT / GAGGTGCGAGCCTTCTGAA / KCNH2 / 150647009 / 150647204
A / CCAGCATGCACCAGTGTGT / GGTCCCAAGGGAGGGTGT / KCNH2 / 150647336 / 150647553
A / TGGTGGAAGCGGATGAACTC / GCTGAGACTGAGACACTGACCT / KCNH2 / 150648091 / 150648310
A / GTGAAGTAGAGCGCCGTCA / GGTACGCCATCGGCAACAT / KCNH2 / 150648628 / 150648778
A / CACTACCTCCCACCACATTCC / TCAACTTCCGCACCACCTAC / KCNH2 / 150649447 / 150649664
A / GTGTGAAGACAGCCGTGTAGAT / GGCAGAGATGTCATCGCTCCT / KCNH2 / 150649794 / 150650017
A / TCCAAGCTCCTCCAAGGTGA / CACCATTAGCAAGATTCCCCAAAT / KCNH2 / 150654331 / 150654523
A / GCACCCAGGACGTAGTGAA / CGCTCCCGAGAAAGCTG / KCNH2 / 150655039 / 150655252
A / TGCCACGTGGTTGTCCAT / ACCACGTGCCTCTCCTCT / KCNH2 / 150655394 / 150655615
A / GGCCCGCTCCTACCATC / GAGAACTGCGCCGTCATC / KCNH2 / 150671786 / 150671997
A / CCCATCCACACTCGGAAGA / CCCATGGGCTCAGGATGC / KCNH2 / 150674855 / 150675015
A / GGGCCTGGCCAAGAAGTG / GGCGCGTGCTGTAGATG / KCNQ1 / 2466412 / 2466620
A / TTTTCGAAGCACTGTCTGTTCCT / CATACTGCTCGATGGTGGACA / KCNQ1 / 2549034 / 2549216
A / GAGCCTGCCTGCAGTGA / GGAAACCTGGGCGTGACC / KCNQ1 / 2591819 / 2592025
A / CCGGCGTGAACAGCTGA / CGTCCTGTCCCCAACCT / KCNQ1 / 2593184 / 2593387
A / CCTCCCGAGGCTCCAGT / GGACCCCAGCTGTCCAA / KCNQ1 / 2604612 / 2604826
A / GTGGCTCAGCAGGTGACA / TCCCCACCTGCTAGCAAGA / KCNQ1 / 2608758 / 2608974
A / GGAGAGAAGATGCTCACAGTCC / GGAGAAACTCACTGGCTTGCT / KCNQ1 / 2609991 / 2610171
A / CAGGGTGGCCACTCACAAT / CCACTATGGGCAGGAGAGAAC / KCNQ1 / 2790032 / 2790245
A / CCCACAGACGACAGTGCATC / GCAATGGCCCATTCTGACATCAT / KCNQ1 / 2798145 / 2798364
A / ACTGACTCTCTCGTCTGCCTT / GTAGGTGGGCAGGGTGTTG / KCNQ1 / 2868965 / 2869188
A / CTGGTTTGTGACTGACTGCATT / ACTTCCTTCCCACCCTCCTATG / SCN5A / 38591730 / 38591952
A / CGCCTGCTGACGGAAGA / GGAGATGGACGCCCTGAAG / SCN5A / 38592067 / 38592265
A / GATACGGAGTGGCTCAGACAG / CTTCCTCATCGTGGTCAACATGTA / SCN5A / 38592382 / 38592586
A / CCCGAGAGCCATTGCTGTT / CATCTTTGGCATGGCCAACTTC / SCN5A / 38592645 / 38592847
A / GTCTGAGGATGCGGCCTATTC / AGACAGAGGTGCCACCAGTA / SCN5A / 38592957 / 38593095
A / TTGGCCAAGATGTTGATTTTCTCA / ACTTTCCATTTGCCTCTCCTTTCAG / SCN5A / 38595898 / 38596065
A / GTTCTTACAGGCCTAGGCATGTTA / CATTGACAACTTCAACCAACAGAAGAAA / SCN5A / 38597743 / 38597962
A / GGCTTTCAGATGCAGACACTGAT / TGTCCAGACCAGAGCCCTAAG / SCN5A / 38598629 / 38598853
A / ACTCACACTGGCTCTTGTTGTTC / GAACTGAGCCACTTCTCTGTCC / SCN5A / 38601727 / 38601948
A / GCAATGGGTTTCTCCTTCCTGTT / AGTATGCCGACAAGATGTTCACA / SCN5A / 38607855 / 38608021
A / GGGTTGAGAGTTTGTGAGCTGTT / CTGGTTCGAGACATTCATCATCTTCA / SCN5A / 38616618 / 38616839
A / GGAGGAGGTGGGCAGATATCTAA / CAGGATGTCAAGGACCCAGAG / SCN5A / 38618023 / 38618188
A / TCAGGGACAAAGGCATGCATT / GTGTGTGGACTCAGCTCATAGG / SCN5A / 38620802 / 38621023
A / CCAGGCTGTTCTCCTCATCTT / GCATTGCCACCCCCTACTC / SCN5A / 38622448 / 38622639
A / CAGCTGGAGGTTGTTCATCTCTC / TTGGTGAGCCTGACCCATTATC / SCN5A / 38622767 / 38622891
A / GGAAGATGATGAGGAAGGCATGAAAG / CAAATCATGGCCCACCCTGA / SCN5A / 38627292 / 38627512
A / GACAGATCAAAGTAGGGATGACCAA / ACATCTTCGACAGCATCATCGTC / SCN5A / 38628789 / 38628982
A / CCACCCTCAAAAGATCCTTCTGT / CACCATCACTATGTGCATCGTACT / SCN5A / 38639095 / 38639319
A / GGTACCTGCAGCGGAAGT / CCCTTTTCCCCAGGCTGA / SCN5A / 38640359 / 38640583
A / CCAGTAATGAGACCACCCCATTG / CCTGGGTTCTGAAGCAGATTTTG / SCN5A / 38645280 / 38645485
A / GCTAGAACCACAGCTGGGAT / CCGTTCCTCCACTCTTTTCCA / SCN5A / 38646197 / 38646421
A / CAGGTTCACCAGGTAGAAGGAC / GGTGACTTGGAAATGCCATAACC / SCN5A / 38647559 / 38647709
A / GGTCAGGGCATAAATAGAAAATACAGTCA / GGATGCAAGGCGGAAACTTT / SCN5A / 38649547 / 38649767
A / CAAGTTCGCACCTGGATCACT / TCTGTGAAGAAGCTGGCTGATG / SCN5A / 38651214 / 38651423
A / CTCCCAGGTGGCTGGAAAG / CTCCTCTGACTGTGTGTCTCCTAT / SCN5A / 38655170 / 38655393
A / CATAGCACAGCATAGCAAATGAGAT / GCTCAGGCCTCCCTAAGAAAC / SCN5A / 38662303 / 38662505
A / GGTACTCAGCAGGTATTAACTGCAA / AGGGCCTAATGCTACTGTCTGT / SCN5A / 38671770 / 38671948
A / GCCATAGAGATCTGGCAGCTT / GCAGGATGAGAAGATGGCAAAC / SCN5A / 38674592 / 38674811
B / GTGTCATTAATCCACCCCTCACC / GTCGTGCTATGTCGTTGAAAACC / KCNE1 / 35821401 / 35821621
B / CATGATGCCCAGGGTGAAGAA / GTGGAACCTTAATGCCCAGGAT / KCNE1 / 35821747 / 35821952
B / AGCCAAAGTTGATGCTGAGAACT / GTGGCCTTCGATTCTTCTAGATTCAA / KCNE2 / 35742891 / 35743103
B / CACGAGGCAAATCCAAATTGTCT / CAAGCAAGCATAAATCACTGAGATTGAA / KCNE2 / 35743208 / 35743342
B / GAAGCAGGGCTGGAGCTT / TGAGCATCCCCCACTTCCT / KCNH2 / 150643945 / 150644168
B / CCCCAGAAGCTGAAAATGTTGGA / CCTGATGGAGGACTGCGA / KCNH2 / 150644563 / 150644743
B / CCCTCATCCTCACTGCTCTCA / CTCTGAGGCCCATTCTCTGTTT / KCNH2 / 150644825 / 150644995
B / GGGACTTTTGTAGGCTGCTCTA / GTGCTGGACATGTACCCTGAG / KCNH2 / 150645798 / 150646015
B / CAGGACTCCCTTTGCTTTGGAT / GTTCCCACATTCTCCTTCCCTTC / KCNH2 / 150646844 / 150647051
B / CAGCCCCAGTGACTGCATA / GAAGTTCAAGACCACACATGCAC / KCNH2 / 150647167 / 150647386
B / GAGACTTGTTTGCTGTGCCAA / CGCTACCACACACAGATGCT / KCNH2 / 150647921 / 150648140
B / CAGCCTCAGTTTCCTCCAACT / CCCCTCCATCAAGGACAAGTATG / KCNH2 / 150648463 / 150648669
B / GAGTCCATGTGTGGCTGCT / GCCCCATCAACGGAATGTG / KCNH2 / 150648739 / 150648961
B / CCACCTCCTCGTTGGCATT / GCTCATCCTGCTGCTGGTC / KCNH2 / 150649623 / 150649834
B / AGGAAGGACCGGGTGTCA / GCTGCAGGGAGCCAAGTC / KCNH2 / 150652461 / 150652613
B / GAGGTCCACAAAGTTGAGGGT / CCTGGTCTCCACTCTCGATCTAT / KCNH2 / 150654478 / 150654675
B / GCGCATGGCCTCGATGT / ATGGACAACCACGTGGCA / KCNH2 / 150655187 / 150655411
B / CCCCTGAGCCTGCCCTAA / GGGCTATGTCCTCCCACT / KCNH2 / 150656627 / 150656848
B / AGAAGCCGTCGTTGCAGTA / GCTGTGTGAGTGGAGAATGTGG / KCNH2 / 150671961 / 150672117
B / CTAGACCCGCGCGTCTC / GCCGGTGGCGATACTCA / KCNQ1 / 2466587 / 2466732
B / GTCTGCCTCATCTTCAGCGT / CTGTATCAGGGCAGGACCAAT / KCNQ1 / 2549174 / 2549385
B / GCTGTGGCGATCACGAAAAG / TTGCATCTGAGCAAGGTGGAT / KCNQ1 / 2592511 / 2592735
B / CTGGAGCCCGACACTGT / CCTGAGCTGCCCCTGTC / KCNQ1 / 2594043 / 2594259
B / CCGGTAACCACGTCCTGAG / ACAATAACAGTGACCAAAATGACAGTGA / KCNQ1 / 2606364 / 2606588
B / CGATGTCCAGGAACCGCTAAT / GGGTCGCACGTGATATGGG / KCNQ1 / 2609901 / 2610031
B / GTTGGGTGGGAGGCCTAAC / GACTCCTTGAGCTCCAGTCC / KCNQ1 / 2683146 / 2683369
B / GGGAGGAGAAGTGATGCGT / CCGGCACCGCTGATCAT / KCNQ1 / 2797154 / 2797364
B / CCAGCACTTGGCCCTGATTT / CTTCACGTTCACACGCAGAC / KCNQ1 / 2799166 / 2799354
B / CGTCGACCCTGAGCTCTTC / GCTCTTCTCTCTGGGCCTTTTA / KCNQ1 / 2869143 / 2869342
B / TGGTGGCTCTAGTGACACTGT / TCTTTGAAGCATGCCTCCTTCC / SCN5A / 38591910 / 38592105
B / CATGAACTTCTCCTCCATCTGGAT / TCCTGTCTGACTTTGCCGATG / SCN5A / 38592223 / 38592425
B / AGTTCTCCAGGATGATGGCAATG / CCTACTGCGACCCCACTCT / SCN5A / 38592540 / 38592686
B / CCAGCCTCCCACTTGACATAA / GACGCTCTTCCGAGTCATCC / SCN5A / 38592803 / 38593006
B / TCTCCGTCCAGCTGACTTGTA / GTGGAGACAGATGACCAAAGTCC / SCN5A / 38595742 / 38595944
B / CCCAGAAGATCCTCCCCACT / GCCTCTGAGAACCCCAGAA / SCN5A / 38597101 / 38597295
B / GAGCTGGTGCTCTACGTATCTT / CTGTGTGATTTCTCCCTTGCAG / SCN5A / 38597913 / 38598091
B / GGGAGGAAGGAAGTCCCTTCT / TGCCTTTGAACTACACCATCGT / SCN5A / 38601604 / 38601771
B / GCCTCCCACTCCCTGGT / GGGAGCTGTTCCCATCCT / SCN5A / 38603866 / 38604072
B / GCATCTCCAGCACGAAGACATA / GCTTCATGTCCACCTTGTCTGG / SCN5A / 38607977 / 38608107
B / GCTCCACTGCTGAGTAGGATCA / TGACCCTCACTCTCTCCCATAG / SCN5A / 38616792 / 38616964
B / GCCATTACCTTCAGTGAAGCAGT / CCCTAAGCTCCTGCAGACTCT / SCN5A / 38618144 / 38618368
B / AGCTCTGGGCCCTGTATATGTA / GAGTCAGACACAGATGACCAAGAAG / SCN5A / 38622368 / 38622493
B / AGGCACCTTCTCCGTCTCT / ACAGCCCCTGATGAGGACA / SCN5A / 38622590 / 38622808
B / GTGGCCCTTGGCCAACTTA / CGCTGGCACATGATGGACTT / SCN5A / 38627162 / 38627337
B / CCCGATGATCTTGATGAGTGTGT / TTTCAGGCAGGAGCTAGAGAGA / SCN5A / 38627470 / 38627623
B / CAGCTCCATGAGGCTAAGGAT / TCCATCCCAAACAATTCCTGCT / SCN5A / 38628939 / 38629096
B / GCGCCATGAAGAGTGTGTTG / ACAGAATCAAACTAACTCATTGGCTGT / SCN5A / 38639276 / 38639493
B / TTTGGACTTGGCACTGGTGAT / CAAAAAGAACAGCACTGTGGACTG / SCN5A / 38645130 / 38645326
B / CGCTGTGCTGTTTTCATCATCTG / AGAGCACATGTCATGGTCGTG / SCN5A / 38645440 / 38645617
B / CCTACAGTCAGGTGAGGGCTTA / TCTTCTTCATGCTTGTCATCTTCCTG / SCN5A / 38647391 / 38647608
B / ACCCTGAGCCCACACTTG / CCCTGACGGCATGGAACAAA / SCN5A / 38648117 / 38648341
B / GTCTAGCCTGGGAAGTCACAA / GGAATCCCTGGACCTTTACCTC / SCN5A / 38651038 / 38651256
B / CAGAAGACTGTGAGGACCATCA / CCCTTATTCTGTCCCCACCTCT / SCN5A / 38651380 / 38651587
B / CAGAGAGAGAGTCACTTGTAGCTGA / GCGGTGGTTCTGCTTTGTAAT / SCN5A / 38655412 / 38655606
B / CTGGACACAAGCCCCCTT / GCCCATGCTGCTCAGCTT / SCN5A / 38663791 / 38664015
B / GCCTCATGAGCCACCCTAA / GACCTGCAGGCCTCCAAA / SCN5A / 38674407 / 38674630
B / GTGCCCCGAGGTAATAGGAA / TCTGTCCCTGGGCATAGAATCA / SCN5A / 38674770 / 38674895

Suppl. table 2.Summary of theAmpliseq-amplicons and coverage details for the SCN5A (NM_198056), KCNH2 (NM_000238), KCNQ1 (NM_000218), KCNE1 (NM_000219), KCNE2 (NM_172201) genes.

Gene / Exon / Nt_Start / Nt_End / Amplicons / Total_Bases / Missed_Bases / Coverage
SCN5A / 28 / 38591811 / 38593049 / 9 / 1,238 / 0 / 100%
SCN5A / 27 / 38595769 / 38596040 / 2 / 271 / 0 / 100%
SCN5A / 27 / 38595865 / 38596040 / 2 / 175 / 0 / 100%
SCN5A / 26 / 38597146 / 38597251 / 1 / 105 / 0 / 100%
SCN5A / 25 / 38597931 / 38598069 / 2 / 138 / 0 / 100%
SCN5A / 24 / 38598721 / 38598775 / 1 / 54 / 0 / 100%
SCN5A / 23 / 38601637 / 38601919 / 2 / 282 / 0 / 100%
SCN5A / 22 / 38603905 / 38604028 / 1 / 123 / 0 / 100%
SCN5A / 21 / 38607899 / 38608073 / 2 / 174 / 0 / 100%
SCN5A / 20 / 38616787 / 38616942 / 2 / 155 / 0 / 100%
SCN5A / 19 / 38618151 / 38618272 / 2 / 121 / 0 / 100%
SCN5A / 18 / 38620824 / 38620983 / 1 / 159 / 0 / 100%
SCN5A / 18 / 38620824 / 38620986 / 1 / 162 / 0 / 100%
SCN5A / 17 / 38622421 / 38622862 / 4 / 441 / 0 / 100%
SCN5A / 16 / 38627181 / 38627532 / 3 / 351 / 0 / 100%
SCN5A / 15 / 38628890 / 38629064 / 2 / 174 / 0 / 100%
SCN5A / 14 / 38639219 / 38639458 / 2 / 239 / 0 / 100%
SCN5A / 13 / 38640408 / 38640541 / 1 / 133 / 0 / 100%
SCN5A / 12 / 38645202 / 38645574 / 3 / 372 / 0 / 100%
SCN5A / 11 / 38646219 / 38646399 / 1 / 180 / 0 / 100%
SCN5A / 10 / 38647441 / 38647639 / 2 / 198 / 0 / 100%
SCN5A / 9 / 38648159 / 38648301 / 1 / 142 / 0 / 100%
SCN5A / 8 / 38649641 / 38649705 / 1 / 64 / 0 / 100%
SCN5A / 7 / 38651224 / 38651455 / 3 / 231 / 0 / 100%
SCN5A / 6* / 38655233 / 38655325 / 1 / 92 / 0 / 100%
SCN5A / 6 / 38655465 / 38655557 / 1 / 92 / 0 / 100%
SCN5A / 5 / 38662333 / 38662462 / 1 / 129 / 0 / 100%
SCN5A / 4 / 38663890 / 38663980 / 1 / 90 / 0 / 100%
SCN5A / 3 / 38671801 / 38671920 / 1 / 119 / 0 / 100%
SCN5A / 2 / 38674525 / 38674798 / 3 / 273 / 0 / 100%

* SCN5A: NM_001160161

Gene / Exon / Nt_Start / Nt_End / Amplicons / Total_Bases / Missed_Bases / Coverage
KCNH2 / 15 / 150642452 / 150642602 / 1 / 150 / 0 / 100%
KCNH2 / 14 / 150643964 / 150644142 / 1 / 178 / 0 / 100%
KCNH2 / 13 / 150644415 / 150644602 / 2 / 187 / 0 / 100%
KCNH2 / 12 / 150644693 / 150644966 / 3 / 273 / 0 / 100%
KCNH2 / 11 / 150645531 / 150645631 / 1 / 100 / 0 / 100%
KCNH2 / 10 / 150645943 / 150646137 / 2 / 194 / 0 / 100%
KCNH2 / 9 / 150647255 / 150647508 / 2 / 253 / 0 / 100%
KCNH2 / 5* / 150646986 / 150647508 / 4 / 522 / 0 / 100%
KCNH2 / 8 / 150648008 / 150648208 / 2 / 200 / 0 / 100%
KCNH2 / 7 / 150648535 / 150648923 / 3 / 388 / 0 / 100%
KCNH2 / 6 / 150649512 / 150649941 / 3 / 429 / 0 / 100%
KCNH2 / 1* / 150652483 / 150652591 / 1 / 108 / 0 / 100%
KCNH2 / 5 / 150654378 / 150654590 / 2 / 212 / 0 / 100%
KCNH2 / 4 / 150655146 / 150655590 / 3 / 444 / 18 / 95.9%
KCNH2 / 3 / 150656659 / 150656824 / 1 / 165 / 0 / 100%
KCNH2 / 2 / 150671798 / 150672029 / 2 / 231 / 4 / 98.3%
KCNH2 / 1 / 150674925 / 150675001 / 1 / 76 / 4 / 94.7%
Gene / Exon / Nt_Start / Nt_End / Amplicons / Total_Bases / Missed_Bases / Coverage
KCNQ1 / 1 / 2466328 / 2466714 / 2 / 386 / 101 / 73.8%
KCNQ1 / 2 / 2549157 / 2549248 / 2 / 91 / 0 / 100%
KCNQ1 / 3 / 2591857 / 2591984 / 1 / 127 / 0 / 100%
KCNQ1 / 4 / 2592554 / 2592633 / 1 / 79 / 0 / 100%
KCNQ1 / 5 / 2593242 / 2593339 / 1 / 97 / 0 / 100%
KCNQ1 / 6 / 2594075 / 2594216 / 1 / 141 / 0 / 100%
KCNQ1 / 7 / 2604664 / 2604775 / 1 / 111 / 0 / 100%
KCNQ1 / 8 / 2606441 / 2606537 / 1 / 96 / 0 / 100%
KCNQ1 / 9 / 2608799 / 2608922 / 1 / 123 / 0 / 100%
KCNQ1 / 10 / 2609942 / 2610084 / 2 / 142 / 0 / 100%
KCNQ1 / 11 / 2683190 / 2683311 / 1 / 121 / 0 / 100%
KCNQ1 / 12 / 2790073 / 2790149 / 1 / 76 / 0 / 100%
KCNQ1 / 13 / 2797189 / 2797284 / 1 / 95 / 0 / 100%
KCNQ1 / 14 / 2798215 / 2798262 / 1 / 47 / 0 / 100%
KCNQ1 / 15 / 2799205 / 2799267 / 1 / 62 / 0 / 100%
KCNQ1 / 16 / 2868996 / 2869233 / 2 / 237 / 0 / 100%
Gene / Exon / Nt_Start / Nt_End / Amplicons / Total_Bases / Missed_Bases / Coverage
KCNE2 / 2 / 35742777 / 35743440 / 5 / 663 / 0 / 100%
Gene / Exon / Nt_Start / Nt_End / Amplicons / Total_Bases / Missed_Bases / Coverage
KCNE1 / 4 / 35821542 / 35821982 / 4 / 440 / 0 / 100%

* KCNH2: NM_001204798

Suppl. table 3. Summary of the Ampliseq for the five genes.

Gene / Chr / HG19 ref seq / Nucleotides / Amplicons / Total
bases / Missed
bases
SCN5A / 3 / NM_198056 (isoform a) NM_001160161 (isoform f) / 38,591,812-38,674,798 / 56 / 6,477 / 0
KCNQ1 / 11 / NM_000218 / 150,642,453-150,675,001 / 20 / 2,031 / 101
KCNH2 / 7 / NM_000238 (isoform a)
NM_001204798 (isoform d) / 2,466,329-2,869,233 / 31 / 4,110 / 26
KCNE1 / 21 / NM_000219 / 38,821,543-38,821,982 / 4 / 440 / 0
KCNE2 / 21 / NM_172201 / 35,742,778-35,743,440 / 5 / 663 / 0

Suppl. table 4. GC content of failure amplicons.

Gene / Amplicon id / Exon / Coverage / GC content (%)
SCN5A / AMPL596505895 / 16 / 1X / 55%
KCNH2 / AMPL2537809643 / 15 / 16X / 69%
KCNH2 / AMPL597723511 / 12 / 1X / 80%
KCNH2 / AMPL597731769 / 11 / 1X / 68%
KCNH2 / AMPL598642619 / 4 / 1X / 75%
KCNH2 / AMPL598642685 / 4 / 23X / 80%
KCNH2 / AMPL598642924 / 4 / 6X / 78%
KCNH2 / AMPL1021467300 / 1 / 1X / 72%
KCNQ1 / AMPL536234459 / 1 / 1X / 82 %
KCNQ1 / AMPL535824760 / 13 / 1X / 66%

Suppl. table5.Summary of the variants identified in the massive sequencing of the five genes in the DNA pool.

gene / nucleotide / Ref
allele / Variant / % variant / TOTAL
READS / SNP / effect
KCNH2 / 150644428 / C / A / 3.66 / 1,329 / rs36210421 / p.R1047>L
KCNH2 / 150644805 / G / A / 3.17 / 2,350 / Novel / p.P952>S
KCNH2 / 150647100 / C / T / 6.2 / 6,383 / rs139247073 / Intron
KCNH2 / 150647969 / C / T / 41.02 / 7,982 / rs41258144 / Intron
KCNH2 / 150647970 / C / G / 36.68 / 9,162 / rs2072412 / Intron
KCNH2 / 150648198 / A / G / 68.19 / 3,423 / rs1137617 / p.Y652Y
KCNH2 / 150648789 / T / C / 46.58 / 8,132 / rs33959111 / p.L564L
KCNH2 / 150649531 / G / A / 30.39 / 11,422 / rs1805120 / p.F513F
KCNH2 / 150649603 / G / A / 36.58 / 7,141 / rs740952 / p.I489I
KCNH2 / 150654622 / A / AG / 3 / 5,277 / rs72573482 / Intron
KCNH2** / 150674932 / C / T / 50 / 20 / Novel / p.Gly24Ser
KCNQ1 / 2549061 / G / C / 4.08 / 5,999 / rs28730663 / Intron
KCNQ1* / 2549329 / TGG / T / 53.25 / 2,183 / Novel / Intron
KCNQ1 / 2608850 / G / T / 3.74 / 8,089 / rs12720457 / p.K393>N
KCNQ1 / 2790163 / T / C / 3.28 / 9,616 / rs11024034 / Intron
KCNQ1** / 2797320 / A / G / 86.11 / 36 / rs163150 / Intron
KCNQ1 / 2798305 / T / C / 61.57 / 10,909 / rs81204 / Intron
KCNQ1 / 2799299 / G / T / 25.0 / 2,090 / rs41282928 / Intron
KCNQ1* / 2869072 / A / AC / 24.63 / 2,100 / Novel / Intron
KCNQ1 / 2869188 / C / T / 11.19 / 4,914 / rs11601907 / p.Y662Y
SCN5A / 38592406 / A / G / 46.06 / 12,900 / rs1805126 / p.D1819D
SCN5A / 38592503 / C / T / 3.43 / 13,362 / rs199473316 / p.S1787>N
SCN5A / 38592734 / G / A / 3.73 / 7,620 / rs 137854604 / p.S1710>L
SCN5A / 38597145 / A / G / 3.06 / 2,245 / Novel / Intron
SCN5A / 38597867 / C / T / 3.4 / 4,546 / rs41315505 / Intron
SCN5A / 38598669 / A / G / 18.77 / 10,184 / rs41312393 / Intron
SCN5A / 38598694 / G / A / 3.45 / 10,523 / rs41312395 / Intron
SCN5A / 38622467 / T / C / 94.01 / 9,579 / rs7430407 / p.E1061E
SCN5A / 38622687 / C / T / 2.5 / 2,005 / Novel / p.R988>Q
SCN5A / 38622727 / G / A / 3.39 / 2,444 / rs41311135 / p.R975>W
SCN5A / 38645420 / T / C / 29.39 / 5,849 / rs1805124 / p.H558>R
SCN5A / 38647642 / G / T / 33.28 / 5,184 / rs41312433 / Intron
SCN5A / 38674475 / C / T / 3.54 / 7,096 / rs41261744 / Intron
SCN5A / 38674712 / T / C / 87.28 / 4,952 / rs6599230 / p.A29A
KCNE1 / 35821821 / T / C / 43.71 / 9,561 / rs1805127 / p.S38>G
KCNE1 / 35821680 / C / T / 2.67 / 7,902 / rs1805128 / p.D85>N
KCNE2 / 35742799 / A / G / 2.91 / 5,883 / rs2234916 / p.T8>A

* False Positive

** Discarded

Suppl. figure 1. Scheme of the procedure to sequence the 20 patients as independent samples or as a single DNA pool. The main difference was the non necessity for individual barcoding of each sample in the pooling approach. This would result in a significant reduction of cost and sample-preparation time when large amounts of individuals are analysed.

Suppl. figure 2. NGS data from amplicons containing two SCN5A and KCNH2 variants, and Sanger electropherogram of the two individuals heterozygous for these nucleotide changes.