Brandon:LabNotes/Project1/2015-9-18: Difference between revisions
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==combinatorial method sequences detail == | ==combinatorial method sequences detail== | ||
*method originally from paper on haplotyping using contiguity preserved transposition [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html#supplementary-information Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing] | |||
*method used in [http://www.sciencemag.org/content/348/6237/910.full#ref-17 Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing] using nuclei instead of haplotyping | |||
*sequence detail for methods used in above papers | |||
ungrouped sequences are nXTv2 i5/i7 sequences | |||
{sequences added by them} | |||
[moasic end Tn5 binding] | |||
(barcode) | |||
p5_barcoded_transposon | |||
5'- TCGTCGGCAGCGTC{TCCACGC}(XXXXXXXX){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ -5' | |||
p7_barcoded_transposon | |||
5'- GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(XXXXXXXX){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ -5' | |||
After single insertion (Tn5 have only i5 or i7 bound, fragments, 9 base pair gap) | |||
p5_barcoded_transposon | |||
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' | |||
NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' | |||
p7_barcoded_transposon | |||
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' | |||
NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' | |||
After tagementation fragment options: | |||
-8 p5 barcodes, 12 p7 barcodes in 8X12 array for 96 unique barcoded fragments. | |||
-Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped. | |||
p5_barcoded_transposon p7_barcoded_transposon | |||
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' | |||
p7_barcoded_transposon p5_barcoded_transposon | |||
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' | |||
p5_barcoded_transposon p5_barcoded_transposon | |||
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' | |||
p7_barcoded_transposon p7_barcoded_transposon | |||
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | |||
3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' | |||
After fill in at 72 for 3-5 minutes (gap filling). | |||
p5_barcoded_transposon p7_barcoded_transposon | |||
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' | |||
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' | |||
p7_barcoded_transposon p5_barcoded_transposon | |||
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3' | |||
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' | |||
p5_barcoded_transposon p5_barcoded_transposon | |||
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3' | |||
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' | |||
p7_barcoded_transposon p7_barcoded_transposon | |||
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' | |||
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' | |||
Now PCRable, adding on dual barcodes. | |||
-8 i5 barcodes, 12 i7 barcodes in 8X12 array for 96 unique barcoded fragments. | |||
-with previously added barcoded fragments makes 9216 unique compartments | |||
i5_adapter | |||
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' | |||
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' | |||
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' | |||
3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' | |||
i7_adapter | |||
Amplification with primer 1 and primer 2 (optional) | |||
primer_1 | |||
5'- AATGATACGGCGACCACCGA -3' | |||
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' | |||
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' | |||
3'- AGCATACGGCAGAAGACGAAC -5 | |||
primer_2 | |||
Read primers for sequencing: | |||
for index reads, long reads, ~37 or more bp, were used for reading both indexes, for each index primer | |||
i5_nXTv2_index_read -----> read_1 -----> index_read_1 -----> | |||
AATGATACGGCGACCACCGAGATCTACAC GCGATCGAGGACGGCAGATGTGTATAAGAGACAG CTGTCTCTTATACACATCTGAGGCGGAGACGGTG | |||
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' | |||
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' | |||
GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC | |||
<----- read_2 | |||
Sequences used: | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT v2] used for i5/i7 barcoding | |||
below sequences from [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html#supplementary-information Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing] | |||
Oligo Sequence (5'-3') | |||
P5_i5_1_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCTATAGCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_2_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_3_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCCCTATCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_4_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCGGCTCTGAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_5_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCAGGCGAAGGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_6_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCTAATCTTAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_7_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCCAGGACGTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P5_i5_8_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
P7_i7_1_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCGAGTAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_2_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTCTCCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_3_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCAATGAGCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_4_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGGAATCTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_5_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCTGAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_6_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCACGAATTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_7_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCAGCTTCAGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_8_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCATTACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_9_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCATAGCCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_10_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCGCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_11_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCGAGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
P7_i7_12_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCTATCGCTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
Read 1 Sequencing Primer (Extended ME A) GCGATCGAGGACGGCAGATGTGTATAAGAGACAG | |||
Read 2 Sequencing Primer (Extended ME B) CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG | |||
Index 1 Sequencing Primer (RC of Extended ME B) CTGTCTCTTATACACATCTGAGGCGGAGACGGTG | |||
pMENTS 5Phos/CTGTCTCTTATACACATCT | |||
NOTES: | |||
i5 universal connector | i5 universal connector | ||
AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer | AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(CTCTCTAT)TCGTCGGCAGCGTC i5 barcode + flowcell adaptor | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(TATCCTCT)TCGTCGGCAGCGTC | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(GTAAGGAG)TCGTCGGCAGCGTC | ||
GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 | GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 | ||
TCGTCGGCAGCGTC{TCCACGC} | TCGTCGGCAGCGTC{TCCACGC}(TATAGCCT){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i5_transposon + bc | ||
TCGTCGGCAGCGTC{TCCACGC} | TCGTCGGCAGCGTC{TCCACGC}(ATAGAGGC){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] | ||
i7 universal connector | i7 universal connector | ||
CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 | CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 | ||
GTCTCGTGGGCTCGG{CTGTCCCTGTCC} | GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(CGAGTAAT){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] | ||
GTCTCGTGGGCTCGG{CTGTCCCTGTCC} | GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(TCTCCGGA){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] | ||
GGCTCGGGTGCTCTG | GGCTCGGGTGCTCTG(ATTCCGCT)TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor | ||
GGCTCGGGTGCTCTG | GGCTCGGGTGCTCTG(GCTCCGAC)TAGAGCATACGGCAGAAGACGAAC | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(TAATGAGC){CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(AGGCCTCT){CCTGTCCCTGTC}GGCTCGGGTGCTCTG | ||
CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1 | CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1 |
Latest revision as of 22:48, 21 September 2015
combinatorial method sequences detail[edit]
- method originally from paper on haplotyping using contiguity preserved transposition Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing
- method used in Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing using nuclei instead of haplotyping
- sequence detail for methods used in above papers
ungrouped sequences are nXTv2 i5/i7 sequences {sequences added by them} [moasic end Tn5 binding] (barcode) p5_barcoded_transposon 5'- TCGTCGGCAGCGTC{TCCACGC}(XXXXXXXX){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' p7_barcoded_transposon 5'- GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(XXXXXXXX){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' After single insertion (Tn5 have only i5 or i7 bound, fragments, 9 base pair gap) p5_barcoded_transposon NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' p7_barcoded_transposon NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' After tagementation fragment options: -8 p5 barcodes, 12 p7 barcodes in 8X12 array for 96 unique barcoded fragments. -Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped. p5_barcoded_transposon p7_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' p7_barcoded_transposon p5_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' p5_barcoded_transposon p5_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' p7_barcoded_transposon p7_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' After fill in at 72 for 3-5 minutes (gap filling). p5_barcoded_transposon p7_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' p7_barcoded_transposon p5_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3' 3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' p5_barcoded_transposon p5_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3' 3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5' p7_barcoded_transposon p7_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' Now PCRable, adding on dual barcodes. -8 i5 barcodes, 12 i7 barcodes in 8X12 array for 96 unique barcoded fragments. -with previously added barcoded fragments makes 9216 unique compartments i5_adapter 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' 5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' i7_adapter Amplification with primer 1 and primer 2 (optional) primer_1 5'- AATGATACGGCGACCACCGA -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- AGCATACGGCAGAAGACGAAC -5 primer_2 Read primers for sequencing: for index reads, long reads, ~37 or more bp, were used for reading both indexes, for each index primer i5_nXTv2_index_read -----> read_1 -----> index_read_1 -----> AATGATACGGCGACCACCGAGATCTACAC GCGATCGAGGACGGCAGATGTGTATAAGAGACAG CTGTCTCTTATACACATCTGAGGCGGAGACGGTG 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC <----- read_2 Sequences used: Nextera XT v2 used for i5/i7 barcoding below sequences from Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing Oligo Sequence (5'-3') P5_i5_1_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCTATAGCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_2_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_3_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCCCTATCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_4_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCGGCTCTGAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_5_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCAGGCGAAGGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_6_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCTAATCTTAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_7_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCCAGGACGTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P5_i5_8_Universal_Connector_A_C15_ME TCGTCGGCAGCGTCTCCACGCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG P7_i7_1_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCGAGTAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_2_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTCTCCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_3_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCAATGAGCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_4_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGGAATCTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_5_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCTGAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_6_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCACGAATTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_7_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCAGCTTCAGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_8_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCATTACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_9_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCATAGCCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_10_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCGCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_11_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCGAGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG P7_i7_12_Universal_Connector_B_D15_ME GTCTCGTGGGCTCGGCTGTCCCTGTCCCTATCGCTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG Read 1 Sequencing Primer (Extended ME A) GCGATCGAGGACGGCAGATGTGTATAAGAGACAG Read 2 Sequencing Primer (Extended ME B) CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG Index 1 Sequencing Primer (RC of Extended ME B) CTGTCTCTTATACACATCTGAGGCGGAGACGGTG pMENTS 5Phos/CTGTCTCTTATACACATCT NOTES: i5 universal connector AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer AATGATACGGCGACCACCGAGATCTACAC(CTCTCTAT)TCGTCGGCAGCGTC i5 barcode + flowcell adaptor AATGATACGGCGACCACCGAGATCTACAC(TATCCTCT)TCGTCGGCAGCGTC AATGATACGGCGACCACCGAGATCTACAC(GTAAGGAG)TCGTCGGCAGCGTC GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 TCGTCGGCAGCGTC{TCCACGC}(TATAGCCT){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i5_transposon + bc TCGTCGGCAGCGTC{TCCACGC}(ATAGAGGC){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i7 universal connector CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(CGAGTAAT){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(TCTCCGGA){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] GGCTCGGGTGCTCTG(ATTCCGCT)TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor GGCTCGGGTGCTCTG(GCTCCGAC)TAGAGCATACGGCAGAAGACGAAC GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(TAATGAGC){CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(AGGCCTCT){CCTGTCCCTGTC}GGCTCGGGTGCTCTG CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1