The Linear Amplification via Transposon Insertion (LIANTI) combines Tn5 transposition and T7 in vitro transcription for linear amplification of genomic DNA from single cells. It does not have the limitaion used in the traditional Tn5 where only fragments with different ends can be amplified. In LIANTI, due to T7 in vitro transcription, every single cutting events are amplifiable. This greatly improves the
LIANTI transposon DNA: 5'- /Phos/
Second strand primer: 5'-
NEBNext Hairpin Adaptor: 5'- /5Phos/
(dU) ACAC TCTTTCCCTACACGAC |GCTCTTCCGATC*T -3' |CGAGAAGGCTAG -5' ----CTGACCTCAAGTCTGCACA
NEBNext Universal PCR Primer for Illumina: 5'-
NEBNext Index 1 Primer for Illumina: 5'-
Illumina TruSeq Read 1 primer: 5'-
Illumina TruSeq Read 2 primer: 5'-
Sample index sequencing primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
CGACTCACTATAGGG GAACAGAATTTAATA |AGATGTGTATAAGAGACAG XXXXXXXXXXXX...XXX 5'-CTGTCTCTTATACACATCT | |TCTACACATATTCTCTGTC -5' XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGA |ATAAT TTAAGACAAGGGGATATCACTCAGC
IVT starts from here ↱ 5'- CTGTCTCTTATACACATCT GAACAGAATTTAATACGACTCACTATAGGG AGATGTGTATAAGAGACAG XXX...XXXCTGTCTCTTATACACATCT CCCTATAGTGAGTCGTATTA AATTCTGTTCAGATGTGTATAAGAGACAG -3' 3'-GACAGAGAATATGTGTAGA CTTGTCTTAAATTATGCTGAGTGATATCCC TCTACACATATTCTCTGTC XXX...XXXGACAGAGAATATGTGTAGA GGGATATCACTCAGCATAAT TTAAGACAAGTCTACACATATTCTCTGTC -5' ↵ IVT starts from here
5'- G AGAUGUGUAUAAGAGACAG XXXXXX...XXXXXXCUGUCUCUUAUACACAUCU CCCUAUAGUGAGUCGUAUUA AAUUCUGUUCAGAUGUGUAUAAGAGACAG -3'
CUUGUCUUAA AUUAU <-----------GACAGAGAAUAUGUGUAGA | 5'-G AGAUGUGUAUAAGAGACAG XXXXXX...XXXXXXCUGUCUCUUAUACACAUCU |CCCUAUAGUGAGUCG
3'- C TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
5'- [8-bp UMI] GGG AGATGTGTATAAGAGACAG ----------> 3'-C TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
5'- [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -3' 3'-[8-bp UMI] CCC TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
5'- [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXA -3' 3'- A[8-bp UMI] CCC TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXX -5'
(dU) ACAC TCTTTCCCTACACGAC ACACGTCTGAACTCCAGTC ---- |GCTCTTCCGATCT [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXAGATCGGAAGAGC | |CGAGAAGGCTAG A[8-bp UMI] CCC TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXTCTAGCCTTCTCG | ----CTGACCTCAAGTCTGCACA CAGCACATCCCTTTCT CACA (dU)
5'- ACAC TCTTTCCCTACACGAC ACACGTCTGAACTCCAGTC -3'GCTCTTCCGATCT [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXAGATCGGAAGAGC CGAGAAGGCTAG A[8-bp UMI] CCC TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXTCTAGCCTTCTCG 3'-CTGACCTCAAGTCTGCACA CAGCACATCCCTTTCT CACA -5'
(i) First round: Top strand: 5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXAGATCGGAAGAGCACACGTCTGAACTCCAGTC -3' <-------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5' Bottom strand: 5'-CAAGCAGAAGACGGCATACGAGAT [i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT ---------------> 3'-CTGACCTCAAGTCTGCACACGAGAAGGCTAG A[8-bp UMI] CCC TCTACACATATTCTCTGTC XXXXXXXXXXXXXXX...XXXXXXXXXXXXXXXTCTAGCCTTCTCGCAGCACATCCCTTTCT CACA -5' (ii) Later rounds of PCR: Product 1: 5'-AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT ------------> 5'-ACAC TCTTTCCCTACACGACGCTCTTCCGATCT [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC [i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA [8-bp UMI] CCC TCTACACATATTCTCTGTC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5' <-------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG [i7]TAGAGCATACGGCAGAAGACGAAC -5' Product 2: 5'-CAAGCAGAAGACGGCATACGAGAT [i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT ---------------> 5'-CAAGCAGAAGACGGCATACGAGAT [i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT [8-bp UMI] GGG AGATGTGTATAAGAGACAG XXX...XXXAGATCGGAAGAGCGTCGTGTAGGGAAAGA GTGT -3' 3'-GTTCGTCTTCTGCCGTATGCTCTA [i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAG A[8-bp UMI] CCC TCTACACATATTCTCTGTC XXX...XXXTCTAGCCTTCTCGCAGCACATCCCTTTCT CACA -5' <-------------------TCTAGCCTTCTCGCAGCACATCCCTTTCT CACATCTAGAGCCACCAGCGGCATAGTAA -5'
Orientation 1: 5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNN GGG AGATGTGTATAAGAGACAG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN CCC TCTACACATATTCTCTGTC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 TruSeq Read 1 8-bp UMI ME gDNATruSeq Read 2 6-bpIllumina P7 i7 index Orientation 2: 5'-AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT XXX...XXXCTGTCTCTTATACACATCT CCC NNNNNNNN AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA XXX...XXXGACAGAGAATATGTGTAGA GGG NNNNNNNN TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 TruSeq Read 1 gDNAME 8-bp UMI TruSeq Read 2 6-bpIllumina P7 i7 index
5'- ACAC TCTTTCCCTACACGACGCTCTTCCGATCT ------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN CCC TCTACACATATTCTCTGTC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNNNN CCC TCTACACATATTCTCTGTC XXX...XXXTCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT NNNNNNNN GGG AGATGTGTATAAGAGACAG XXX...XXXAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <------------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'