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Drop-seq.html
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<!DOCTYPE html>
<html>
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s5 {color:#6baed6;}
s7 {color:#fc9272;}
p5 {color:#08519c;}
p7 {color:#a50f15;}
me {color:#969696;}
tso {color:#2ca25f;}
cbc {color:#f768a1;}
umi {color:#807dba;}
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<head>
<title>Drop-seq</title>
</head>
<body>
<h1><a href="http://www.cell.com/abstract/S0092-8674(15)00549-8" target="_blank"><span style="color:red;">Drop-seq</span></a> / <a href="https://www.nature.com/nmeth/journal/v14/n4/full/nmeth.4179.html" target="_blank"><span style="color:red;">Seq-Well</span></a></h1>
<p><span style="font-family:verdana; font-size:1.1em">In the original pulication in Cell 161, 1202-1214 (2015), there are two batches of beads, with only two base pairs difference. Here, Beads-oligo-dT-seqA was used as demonstration. Seq-Well used exact the same oligo design with Drop-seq with Beads-oligo-dT-seqB, which was published in Nature Methods 14, 395–398 (2017).</span></p>
<br></br>
<h2>Adapter and primer sequences:</h2>
<seq>
<p>Beads-oligo-dT-seqA: |--5'- TTTTTTT<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'</p>
<p>Beads-oligo-dT-seqB: |--5'- TTTTTTT<tso>AAGCAGTGGTATCAACGCAGAGT</tso>AC<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'</p>
<p>Template Switching Oligo (TSO): 5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>GAATrGrGrG -3'</p>
<p>ISPCR: 5′- <tso>AAGCAGTGGTATCAACGCAGAGT</tso> -3′</p>
<p>Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'- <me>AGATGTGTATAAGAGACAG</me> -3'</p>
<p>Nextera N/S5xx primer entry point (s5): 5'- <s5>TCGTCGGCAGCGTC</s5> -3'</p>
<p>Nextera N7xx primer entry point (s7): 5'- <s7>GTCTCGTGGGCTCGG</s7> -3'</p>
<p>Illumina P5 adapter: 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5> -3'</p>
<p>Illumina P7 adapter: 5'- <p7>CAAGCAGAAGACGGCATACGAGAT</p7> -3'</p>
<p>Library PCR primer 1: 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>AC -3'</p>
<p>Library PCR primer 2 (this is basically Nextera N7xx): 5'- <p7>CAAGCAGAAGACGGCATACGAGAT</p7>[8-bp i7 index]<s7>GTCTCGTGGGCTCGG</s7> -3'</p>
<p>Read 1 sequencing primer (seqA): 5'- GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT -3'</p>
<p>Read 1 sequencing primer (seqB): 5'- GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>AC -3'</p>
<p>Read 2 sequencing primer: 5'- <s7>GTCTCGTGGGCTCGG</s7><me>AGATGTGTATAAGAGACAG</me> -3'</p>
<p>i7 index sequencing primer: 5'- <me>CTGTCTCTTATACACATCT</me><s7>CCGAGCCCACGAGAC</s7> -3'</p>
</seq>
<br></br>
<h2>Step-by-step library generation</h2>
<h3>(1) mRNA capture using Beads-oligo-dT in the droplets:</h3>
<pre>
<seq>
5'- XXXXXXXXXXXXXXXXXXXB(A)<sub>n</sub>
<----NV(T)<sub>30</sub><umi>[8-bp UMI]</umi><cbc>[12-bp cell barcode]</cbc>TGCA<tso>TGAGACGCAACTATGGTGACGAA</tso>TTTTTTTT -5'--|
</seq>
</pre>
<h3>(2) Break droplets (i.e. droplets are merely used as a cell capture chamber), reverse transcription for all cells in one reaction, the terminal tranferase acitivity of MMLV adds extra Cs:</h3>
<pre>
<seq>
5'- XXXXXXXXXXXXXXXXXXXB(A)<sub>n</sub>
CCCXXXXXXXXXXXXXXXXXXNV(T)<sub>30</sub><umi>[8-bp UMI]</umi><cbc>[12-bp cell barcode]</cbc>TGCA<tso>TGAGACGCAACTATGGTGACGAA</tso>TTTTTTTT -5'--|
</seq>
</pre>
<h3>(3) Adding TSO for second strand synthesis:</h3>
<pre>
<seq>
5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>GAATGGGXXXXXXXXXXXXXXXXXXXX(A)<sub>n</sub>---->
<------CCCXXXXXXXXXXXXXXXXXXXX(T)<sub>30</sub><umi>[8-bp UMI]</umi><cbc>[12-bp cell barcode]</cbc>TGCA<tso>TGAGACGCAACTATGGTGACGAA</tso>TTTTTTTT -5'--|
</seq>
</pre>
<h3>(4) Adding ISPCR for single primer cDNA amplification:<a href="http://www.nature.com/nmeth/journal/v7/n7/full/nmeth.1470.html" target="_blank">( i.e. semi-suppressive PCR )</a></h3>
<pre>
<seq>
5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>---->
5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>GAATGGGXXXXX...XXXXX(pA)<umi>[8-bp UMI]</umi><cbc>[12-bp cell barcode]</cbc>ACGT<tso>ACTCTGCGTTGATACCACTGCTT</tso>AAAAAAAA
<tso>TTCGTCACCATAGTTGCGTCTCA</tso>CTTACCCXXXXX...XXXXX(dT)<umi>[8-bp UMI]</umi><cbc>[12-bp cell barcode]</cbc>TGCA<tso>TGAGACGCAACTATGGTGACGAA</tso>TTTTTTTT -5'--|
<----<tso>TGAGACGCAACTATGGTGACGAA</tso> -5'
</seq>
</pre>
<h3>(5) Nextera tagmentation on amplified cDNA (will create 9-bp gap):</h3>
<img src="data/tn5_dimer.svg" alt="Tn5 dimer" style="width:800px;height:450px;">
<pre>
<seq>
<i>Product 1 (5'-end of cDNA, with s5 sequence, not amplifiable due to the use of Nextera N7xx for library amplification, see step 6):</i>
5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>GAATGGGXXX...XXX <me>CTGTCTCTTATACACATCT</me>
<tso>TTCGTCACCATAGTTGCGTCTCA</tso>CTTACCCXXX...XXXXXXXXXXXX<me>GACAGAGAATATGTGTAGA</me><s5>CTGCGACGGCTGCT</s5> -5'
<i>Product 2 (5'-end of cDNA, with s7 sequence, not amplifiable due to Library PCR Primer 1 ends with "AC" which cannot to be annealed to the template, see step 6):</i>
5'- <tso>AAGCAGTGGTATCAACGCAGAGT</tso>GAATGGGXXX...XXX <me>CTGTCTCTTATACACATCT</me>
<tso>TTCGTCACCATAGTTGCGTCTCA</tso>CTTACCCXXX...XXXXXXXXXXXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7> -5'
<i>Product 3 (3'-end of cDNA, with s5 sequence, not amplifiable due to the use of Nextera N7xx for library amplification, see step 6):</i>
|--5'- TTTTTTTT<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(dT)XXX...XXX <me>CTGTCTCTTATACACATCT</me>
AAAAAAAA<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(pA)XXX...XXXXXXXXXXXX<me>GACAGAGAATATGTGTAGA</me><s5>CTGCGACGGCTGCT</s5> -5'
<i>Product 4 (3'-end of cDNA, with s7 sequence, the only amplifiable fragment which will be used for library prep and sequencing, see step 6):</i>
|--5'- TTTTTTTT<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(dT)XXX...XXX <me>CTGTCTCTTATACACATCT</me>
AAAAAAAA<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(pA)XXX...XXXXXXXXXXXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7> -5'
</seq>
</pre>
<h3>(6) Add Library PCR Primer 1 & 2 to amplify library:</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>AC-------->
|--5'- TTTTTTTT<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(dT)XXX...XXX <me>CTGTCTCTTATACACATCT</me>
AAAAAAAA<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>[12-bp cell barcode]</cbc><umi>[8-bp UMI]</umi>(pA)XXX...XXXXXXXXXXXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7> -5'
<--------<s7>GGCTCGGGTGCTCTG</s7>[i7]<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(7) Final library structure:</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>NNNNNNNNNNNN</cbc><umi>NNNNNNNN</umi>(dT)XXX...XXX<me>CTGTCTCTTATACACATCT</me><s7>CCGAGCCCACGAGAC</s7>NNNNNNNN<p7>ATCTCGTATGCCGTCTTCTGCTTG</p7>
<p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CGGACAGGCGCC<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>NNNNNNNNNNNN</cbc><umi>NNNNNNNN</umi>(pA)XXX...XXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7>NNNNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
<p5>Illumina P5</p5> <tso>ISPCR/TSO</tso> <cbc>12bp cell</cbc> <umi>8bp</umi> cDNA <me>ME</me> <s7>s7</s7> i7 <p7>Illumina P7</p7>
<cbc>barcode</cbc> <umi>UMI</umi>
</align>
</pre>
<br></br>
<h2>Library sequencing:</h2>
<h3>(1) Add Read 1 sequencing primer (seqA) to sequence the first read (bottom strand as template, sequence 12-bp cell barcode and 8-bp UMI):</h3>
<pre>
<align class="long">
5'- GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT------------------->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CGGACAGGCGCC<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>NNNNNNNNNNNN</cbc><umi>NNNNNNNN</umi>(pA)XXX...XXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7>NNNNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(2) Add i7 index sequencing primer to sequence the i7 index (bottom strand as template):</h3>
<pre>
<align class="long">
5'- <me>CTGTCTCTTATACACATCT</me><s7>CCGAGCCCACGAGAC</s7>------->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CGGACAGGCGCC<tso>TTCGTCACCATAGTTGCGTCTCA</tso>TGCA<cbc>NNNNNNNNNNNN</cbc><umi>NNNNNNNN</umi>(pA)XXX...XXX<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7>NNNNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(3) Cluster regeneration, add Read 2 sequencing primer to sequence the second read (top strand as template, sequence cDNA):</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GCCTGTCCGCGG<tso>AAGCAGTGGTATCAACGCAGAGT</tso>ACGT<cbc>NNNNNNNNNNNN</cbc><umi>NNNNNNNN</umi>(dT)XXX...XXX<me>CTGTCTCTTATACACATCT</me><s7>CCGAGCCCACGAGAC</s7>NNNNNNNN<p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3'
<-------<me>GACAGAGAATATGTGTAGA</me><s7>GGCTCGGGTGCTCTG</s7> -5'
</align>
</pre>
</body>
</html>