NTT-seq library structure and sequencing

NTT-seq main website

Adapter and primer sequences:

Barcoded MEDSA: 5'- TCGTCGGCAGCGTC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'

Standard MEDSB: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Tn5 binding site 19-bp Mosaic End (ME) bottom: 5'- /Phos/CTGTCTCTTATACACATCT -3'

P5 index primer entry point (s5): 5'- TCGTCGGCAGCGTC -3'

P7 index primer entry point (s7): 5'- GTCTCGTGGGCTCGG -3'

P5 index primer: 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3'

P7 index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG -3'

Custom Read 1 sequencing primer: 5'- GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'

Standard Index 1 sequencing primer (i7): 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'

Standard Read 2 seuquencing primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Custom Index 2 sequencing primer (i5): 5'-CTGTCTCTTATACACATCTGCCGTCCTCGATCGC-3'

Step-by-step library generation

(1) Tagmentation leading to sequencable fragment:

 
 5'- TCGTCGGCAGCGTC[8-bp Tn5 index]GCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                                                  TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(2) Linear amplification cycle 1:

Single extension round finishes both strands of the molecule

 
 5'- TCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
 3'- AGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(3) Linear amplification cycle 2:

Linear amplification using a primer at the S5 end only, adding the cell barcode

 
  5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC------>
                                       3'- AGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(4) Library ampification using indexed P5/P7 primers:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC------>
                                     5'- TCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXXXXXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                                     3'- AGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXXXXXXXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                        <------GGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(5) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5            16 bp       s5        8 bp                          ME             gDNA               ME              s7          i7          Illumina P7
                               Cell barcode           Tn5 barcode                                                                              Sample barcode

Library sequencing:

(1) Add custom read 1 sequencing primer to sequence the first read (bottom strand as template, these are the gDNA reads):


                                                            5'- GCGATCGAGGACGGCAGATGTGTATAAGAGACAG------------->
 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGANNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add standard index 1 sequencing primer to sequence the i7 index:


                                                                                                             5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-------->
 3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) NovaSeq: folds over and sequence the second index (Tn5 barcode and cell barcode):


5'- AATGATACGGCGACCACCGAGATCTACAC------------------------------>
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGNNNNNNNNCGCTAGCTCCTGCCGTCTACACATATTCTCTGTCXXXXXX...XXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(4) Cluster regeneration


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'

(5) NextSeq: add custom Index 2 sequencing primer to sequence second Tn5 barcode and i5 index:


                                <------------------------------CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC -5' 
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'

(6) Add standard Read 2 sequencing primer:


                                                                                                 <--------------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' 
5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCNNNNNNNNGCGATCGAGGACGGCAGATGTGTATAAGAGACAGXXXXXX...XXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'