Brandon:LabNotes/Project1/2015-9-21
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combinatorial sequencing adaptation to THS-seq protocol and sequence detail
- adapting THS-seq to combinatorial method sequences detail, however will use 3 barcodes since initial transposition only inserts 1 barcode, and barcode will be 6 base pairs to save on transposon length.
- T7R1-Nextera XT V2 TOP was already validated on Update of THS-seq protocol sequences to use Nextera XT V2 sequences, which adatped from Nextera XT V2 sequences detailed Nextera XT V2 sequences]
Original T7R1-Nextera XT V2 TOP (64 bp) 5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (T7-nXTv2_i5-1-top) \---/ \-/\---------------------/ \--------------------------------/ spcr T7 rec. T7 req. forward read seq. \-----------------/ ME scT7R1-nXTv2_top (74 bp) 5 basepair spacer removed from 5' end barcode (r5) and {TCCACGCGC} added in, was new sequence added in on combinatorial method, TCCACGC[r5]GC originally 5'- [AATTAATACGACTCACTATAGGGAGA]{TCCACGCGC}(XXXXXX)TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’ (scT7_r5XXX_i5_top) \-/\---------------------/ \--------------------------------/ T7 rec. T7 req. forward read seq. \-----------------/ ME Nextera XT V2 Bottom (19 bp with /5Phos/) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) T7 description: +1 [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA] [end sequence] rec. T7 req --> [front sequence for optimal binding] After Annealing: 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (scT7_r5XXX_i5_top) 3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot) 3'- GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' (scT7_r5XXX_i5_top) Fragments created after transposition: 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom: 5'- NNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3’ 3'- NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN -5' After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also) 5'- [AATTAATACGACTCACTATAGGGAGA]TCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' 3'- [TTAATTATGCTGAGTGATATCCCTCT]AGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNNN -5' 5'- NNNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[r5]GCGCGTGGA[TCTCCCTATAGTGAGTCGTATTAATT] -3' 3'- NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCT[AGAGGGATATCACTCAGCATAATTAA] -5' After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT. First G in GGG is the +1 site for the beginning of RNA synthesis for T7. for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x) DNA 5'- GGGAGATCCACGCGC -3' (Tm=57C, anneal at 52C) (sss_scnXTv2) cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5' Now have double stranded DNA, which should be long. DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'
Custom Nextera i7 transposome i7 adapter 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' (nXTv2_i7_top) 3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot) 5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-nXTv2_i7_bot) 3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top) After custom nextera tagmentation: DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' After fill in: DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' Amplification (to add i5/i7 barcodes): for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator) 'All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix" 5'- AATGATACGGCGACCACCGA -3' (i5_PCR_ILAdptr, Tm=59C) 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC -3' (scT7_S5XX, comp region Tm=57C, whole~75C) DNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (i7XX_orig, comp region Tm=57C, whole~71C) 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7XX_long, comp region Tm=59C, whole~71C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' (i7_PCR_ILAdptr, Tm=58C) 3'- AGCATACGGCAGAAGACGAAC -5' Final sequencing ready fragment nXTv2_i5_index_read -----> nXTv2_read1 -----> nXTv2_i7_index_read -----> 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5' 3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' <----- r5_index_read <----- nXTv2_read2
Sequences to order scT7_r5XXX_i5_top Barcoded Transposons P-T7tspn-bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' sss_scnXTv2 5'- GGGAGATCCACGCGC -3' nXTv2_i7_top 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' P-nXTv2_i7_bot 5'- /5Phos/CTGTCTCTTATACACATCT -3' scT7_S5XX Modified Nextera XT V2 i5 barcode sequences and adaptors i7XX_long Extended Nextera XT V2 i7 barcode sequences and adaptors i5_PCR_ILAdptr 5'- AATGATACGGCGACCACCGA -3' i7_PCR_ILAdptr 5'- CAAGCAGAAGACGGCATACGA-3' nXTv2_read1 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' nXTv2_i7_index_read 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' nXTv2_i5_index_read 5'- AATGATACGGCGACCACCGAGATCTACAC -3' r5_index_read 5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3' nXTv2_read2 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
Barcoded scT7_r5XXX_i5_top transposons 74 bp long, using truncated barcodes from Quantitative single-cell RNA-seq with unique molecular identifiers num. r5_barcode r5_smpl_sheet name r5_full_sequence_(5'_->_3') 1 TCTAAT ATTAGA scT7_r5001_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 2 ACTCGT ACGAGT scT7_r5002_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 3 ACGTCA TGACGT scT7_r5003_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 4 AACGAC GTCGTT scT7_r5004_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 5 TTAGCG CGCTAA scT7_r5005_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 6 ACAGAC GTCTGT scT7_r5006_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 7 CGTAGG CCTACG scT7_r5007_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 8 GAGCTC GAGCTC scT7_r5008_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 9 AATACC GGTATT scT7_r5009_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAATACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 10 TCACAC GTGTGA scT7_r5010_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 11 CTGGTA TACCAG scT7_r5011_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 12 GATAGG CCTATC scT7_r5012_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 13 TGTCGA TCGACA scT7_r5013_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 14 ACGGGT ACCCGT scT7_r5014_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 15 CAGCAT ATGCTG scT7_r5015_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 16 TTGACA TGTCAA scT7_r5016_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 17 TTCAAG CTTGAA scT7_r5017_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 18 GTCCTG CAGGAC scT7_r5018_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 19 CACTGA TCAGTG scT7_r5019_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 20 TACAGG CCTGTA scT7_r5020_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 21 ACCTTT AAAGGT scT7_r5021_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 22 CCGTTC GAACGG scT7_r5022_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 23 TGTTTG CAAACA scT7_r5023_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 24 GTGGCT AGCCAC scT7_r5024_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 25 GTACAA TTGTAC scT7_r5025_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 26 CAAAGT ACTTTG scT7_r5026_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 27 GCATGA TCATGC scT7_r5027_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 28 CATTAG CTAATG scT7_r5028_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 29 CTCCTT AAGGAG scT7_r5029_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 30 ACCCGA TCGGGT scT7_r5030_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 31 TGCCAC GTGGCA scT7_r5031_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 32 TACCAA TTGGTA scT7_r5032_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 33 CGAGGT ACCTCG scT7_r5033_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 34 CCACCA TGGTGG scT7_r5034_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 35 TAATCA TGATTA scT7_r5035_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 36 AGTCCA TGGACT scT7_r5036_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 37 CTCAGC GCTGAG scT7_r5037_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 38 TGGACT AGTCCA scT7_r5038_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 39 CAAACC GGTTTG scT7_r5039_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 40 ACTCCG CGGAGT scT7_r5040_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 41 ACCGTG CACGGT scT7_r5041_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 42 AGAACG CGTTCT scT7_r5042_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 43 ATTGAC GTCAAT scT7_r5043_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 44 GTATCC GGATAC scT7_r5044_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 45 CGGAAA TTTCCG scT7_r5045_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 46 TTGTGT ACACAA scT7_r5046_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 47 GCACAT ATGTGC scT7_r5047_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 48 GTCATT AATGAC scT7_r5048_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 49 CCTATA TATAGG scT7_r5049_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 50 ACGCTG CAGCGT scT7_r5050_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 51 GATTGT ACAATC scT7_r5051_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 52 ATGTAG CTACAT scT7_r5052_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 53 CGCTGT ACAGCG scT7_r5053_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 54 AGACTT AAGTCT scT7_r5054_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 55 TGGGCA TGCCCA scT7_r5055_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 56 ACTAGA TCTAGT scT7_r5056_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACTAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 57 ATGGGA TCCCAT scT7_r5057_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 58 GCGACG CGTCGC scT7_r5058_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 59 GGGTAG CTACCC scT7_r5059_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 60 GGTGAA TTCACC scT7_r5060_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 61 CATCTT AAGATG scT7_r5061_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 62 ATAATC GATTAT scT7_r5062_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 63 TATGGT ACCATA scT7_r5063_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTATGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 64 GACTTA TAAGTC scT7_r5064_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 65 TGATTC GAATCA scT7_r5065_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 66 TAGCGA TCGCTA scT7_r5066_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 67 AACTCC GGAGTT scT7_r5067_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 68 GAAGTG CACTTC scT7_r5068_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 69 GAGTAC GTACTC scT7_r5069_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 70 CGCAAT ATTGCG scT7_r5070_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 71 CTGGAG CTCCAG scT7_r5071_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 72 TGGGTT AACCCA scT7_r5072_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 73 TCCTAG CTAGGA scT7_r5073_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 74 TCATGC GCATGA scT7_r5074_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 75 TTCCCT AGGGAA scT7_r5075_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 76 GTAAAG CTTTAC scT7_r5076_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 77 ACAACT AGTTGT scT7_r5077_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCACAACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 78 AGGCAT ATGCCT scT7_r5078_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 79 TGTGTC GACACA scT7_r5079_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 80 AGCATA TATGCT scT7_r5080_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 81 AGACGC GCGTCT scT7_r5081_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 82 CTTGCT AGCAAG scT7_r5082_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 83 TCTGCC GGCAGA scT7_r5083_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 84 TAGTCG CGACTA scT7_r5084_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 85 TTGCTG CAGCAA scT7_r5085_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 86 GACAAC GTTGTC scT7_r5086_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGACAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 87 ATTCCC GGGAAT scT7_r5087_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 88 TTAAGC GCTTAA scT7_r5088_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 89 CGCCTC GAGGCG scT7_r5089_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 90 CTTATC GATAAG scT7_r5090_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 91 CGCTAA TTAGCG scT7_r5091_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 92 AGGTCC GGACCT scT7_r5092_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 93 AAGGTG CACCTT scT7_r5093_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 94 GTTGTA TACAAC scT7_r5094_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 95 AGAGAA TTCTCT scT7_r5095_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 96 ATTTGG CCAAAT scT7_r5096_i5_top AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Modified Nextera XT V2 i5 barcode sequences and adaptors same i7 adaptors/barcodes are used as in original Nextera XT V2 system or 2 bp longer ones can be used as listed below num. i5_barcode i5_smplsheet scT7_i5_name scT7_i5_full_sequence_(5'_->_3') 1 CTCTCTAT CTCTCTAT scT7_S502 AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCCACGCGC 2 TATCCTCT TATCCTCT scT7_S503 AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCCACGCGC 3 GTAAGGAG GTAAGGAG scT7_S505 AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCCACGCGC 4 ACTGCATA ACTGCATA scT7_S506 AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCCACGCGC 5 AAGGAGTA AAGGAGTA scT7_S507 AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCCACGCGC 6 CTAAGCCT CTAAGCCT scT7_S508 AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCCACGCGC 7 CGTCTAAT CGTCTAAT scT7_S510 AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCCACGCGC 8 TCTCTCCG TCTCTCCG scT7_S511 AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCCACGCGC 9 TCGACTAG TCGACTAG scT7_S513 AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCCACGCGC 10 TTCTAGCT TTCTAGCT scT7_S515 AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCCACGCGC 11 CCTAGAGT CCTAGAGT scT7_S516 AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCCACGCGC 12 GCGTAAGA GCGTAAGA scT7_S517 AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCCACGCGC 13 CTATTAAG CTATTAAG scT7_S518 AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCCACGCGC 14 AAGGCTAT AAGGCTAT scT7_S520 AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCCACGCGC 15 GAGCCTTA GAGCCTTA scT7_S521 AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCCACGCGC 16 TTATGCGA TTATGCGA scT7_S522 AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCCACGCGC num. i7_barcode i7_name i7_smplsheet i7_full_sequence_(5'_->_3') 1 TCGCCTTA N701_long TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG 2 CTAGTACG N702_long CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG 3 TTCTGCCT N703_long AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG 4 GCTCAGGA N704_long TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG 5 AGGAGTCC N705_long GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAG 6 CATGCCTA N706_long TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAG 7 GTAGAGAG N707_long CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAG 8 CAGCCTCG N710_long CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAG 9 TGCCTCTT N711_long AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAG 10 TCCTCTAC N712_long GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAG 11 TCATGAGC N714_long GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGGAG 12 CCTGAGAT N715_long ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGGAG 13 TAGCGAGT N716_long ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGGAG 14 GTAGCTCC N718_long GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGGAG 15 TACTACGC N719_long GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGGAG 16 AGGCTCCG N720_long CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGGAG 17 GCAGCGTA N721_long TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGGAG 18 CTGCGCAT N722_long ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGGAG 19 GAGCGCTA N723_long TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGGAG 20 CGCTCAGT N724_long ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGGAG 21 GTCTTAGG N726_long CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGGAG 22 ACTGATCG N727_long CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGGAG 23 TAGCTGCA N728_long TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGGAG 24 GACGTCGA N729_long TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGGAG
original Nextera XT V2 i5/i7 barcode sequences and primers num. i5_barcode i5_name i5_smplsheet i5_full_sequence_(5'_->_3') 1 CTCTCTAT S502 CTCTCTAT AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC 2 TATCCTCT S503 TATCCTCT AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC 3 GTAAGGAG S505 GTAAGGAG AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC 4 ACTGCATA S506 ACTGCATA AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC 5 AAGGAGTA S507 AAGGAGTA AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC 6 CTAAGCCT S508 CTAAGCCT AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC 7 CGTCTAAT S510 CGTCTAAT AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC 8 TCTCTCCG S511 TCTCTCCG AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC 9 TCGACTAG S513 TCGACTAG AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC 10 TTCTAGCT S515 TTCTAGCT AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC 11 CCTAGAGT S516 CCTAGAGT AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC 12 GCGTAAGA S517 GCGTAAGA AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC 13 CTATTAAG S518 CTATTAAG AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC 14 AAGGCTAT S520 AAGGCTAT AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC 15 GAGCCTTA S521 GAGCCTTA AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC 16 TTATGCGA S522 TTATGCGA AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC num. i7_barcode i7_name i7_smplsheet i7_full_sequence_(5'_->_3') 1 TCGCCTTA N701 TAAGGCGA CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG 2 CTAGTACG N702 CGTACTAG CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG 3 TTCTGCCT N703 AGGCAGAA CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG 4 GCTCAGGA N704 TCCTGAGC CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG 5 AGGAGTCC N705 GGACTCCT CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG 6 CATGCCTA N706 TAGGCATG CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG 7 GTAGAGAG N707 CTCTCTAC CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG 8 CAGCCTCG N710 CGAGGCTG CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG 9 TGCCTCTT N711 AAGAGGCA CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG 10 TCCTCTAC N712 GTAGAGGA CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG 11 TCATGAGC N714 GCTCATGA CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG 12 CCTGAGAT N715 ATCTCAGG CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG 13 TAGCGAGT N716 ACTCGCTA CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG 14 GTAGCTCC N718 GGAGCTAC CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG 15 TACTACGC N719 GCGTAGTA CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG 16 AGGCTCCG N720 CGGAGCCT CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG 17 GCAGCGTA N721 TACGCTGC CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG 18 CTGCGCAT N722 ATGCGCAG CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG 19 GAGCGCTA N723 TAGCGCTC CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG 20 CGCTCAGT N724 ACTGAGCG CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG 21 GTCTTAGG N726 CCTAAGAC CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG 22 ACTGATCG N727 CGATCAGT CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG 23 TAGCTGCA N728 TGCAGCTA CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG 24 GACGTCGA N729 TCGACGTC CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG