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<!DOCTYPE html>
<html>
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<head>
<title>inDrop</title>
</head>
<body>
<h1><a href="http://www.cell.com/cell/abstract/S0092-8674(15)00500-0" target="_blank">inDrop</a></h1>
<p><span style="font-family:verdana; font-size:1.1em;">inDrop was originally pulished in Cell 161, 1187–1201 (2015). Then, two years after that, the authors published the detailed protocol in <a href="https://www.nature.com/articles/nprot.2016.154" target="_blank">Nature Protocols 12, 44–73 (2017)</a>, which has different primer design comparing to the orginal paper. The workflow shown here is based on the Nature Protocol paper, which seems to be the version 2 of the inDrop technology. According to the <a href="https://github.com/indrops/indrops" target="_blank">inDrop github page</a>, there is a version 3, but the oligos and library structures are exactly the same as version 2, except the sequencing mode has changed. In version 2, three different reads are generated; in version 3, four different reads are generated. See details below.</span></p>
<br></br>
<h2>Adapter and primer sequences:</h2>
<h3>Sequence used during the experiment:</h3>
<seq>
<p><sup>*</sup>Beads-oligo-dT19V: |--5'- /5Acryd/iSpPC/CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>TTTTTTTTTTTTTTTTTTV -3'</p>
<p>T7 promoter: 5'- <t7>TAATACGACTCACTATAGGG</t7> -3'</p>
<p>PE1 adapter: 5'- <pe1>CTCTTTCCCTACACGACGCTCTTC</pe1> -3'</p>
<p>PE2 adapter: 5'- <pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2> -3'</p>
<p>PE2-N6 RT primer: 5'- <pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>NNNNNN -3'</p>
<p>W1 sequence: 5'- <w1>AAGGCGTCACAAGCAATCACTC</w1> -3'</p>
<p>PE1 PCR primer: 5'- <p7>CAAGCAGAAGACGGCATACGAGAT</p7>[6-bp sample index]<pe1>CTCTTTCCCTACACGA</pe1> -3'</p>
<p>PE2 PCR primer: 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GGTC<pe2>TCGGCATTCCTGCTGAAC</pe2> -3'</p>
<p>Illumina P5 adapter: 5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5> -3'</p>
<p>Illumina P7 adapter: 5'- <p7>CAAGCAGAAGACGGCATACGAGAT</p7> -3'</p>
<p>Read 1 sequencing primer: 5'- <pe2>GGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2> -3'</p>
<p>Index sequencing primer: 5'- AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1> -3'</p>
<p>Read 2 sequencing primer: 5'- <pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT -3'</p>
<p><b><span style="color:red">*</span></b> barcode1 has variable length.</p>
</seq>
<h3>Sequence used for barcoded oligo-dT19V generation (before the experiment):</h3>
<seq>
<p>Acrydite-modified primer: 5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1> -3'</p>
<p>BA19 oligo: 5'- BAAAAAAAAAAAAAAAAAAA -3'</p>
<p>FAM-PE1 probe: 5'-/6-FAM/ AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1> -3'</p>
<p>FAM-W1 probe: 5'-/6-FAM/ <w1>AAGGCGTCACAAGCAATCACTC</w1> -3'</p>
<p>FAM-BA19 probe: 5'-/6-FAM/ BAAAAAAAAAAAAAAAAAAA -3'</p>
<p>Barcode 1 plates (384 of them in 384 individual wells): 5'- <w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>[barcode1]</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1> -3', for the sequences of 384 different barcode1, see <a href="../data/inDrop_barcode1_list.txt" target="_blank">inDrop_barcode1_list.txt</a>.</p>
<p>Barcode 2 plates (384 of them in 384 individual wells): 5'- BAAAAAAAAAAAAAAAAAAA<umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1> -3', for the sequences of 384 different barcode2 (8 bp), see <a href="../data/inDrop_barcode2_list.txt" target="_blank">inDrop_barcode2_list.txt</a>.</p>
</seq>
<br></br>
<h2>Step-by-step generation of oligo-dT19V:</h2>
<h3>(1) Bind Acrydite-modified primer to acrylimide gel beads by mixing:</h3>
<pre>
<seq>
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1> -3'
</seq>
</pre>
<h3>(2) Split the gel beads with bound primers into wells in Barcode 1 plates, and primer extension:</h3>
<pre>
<seq>
<--------<pe1>GAGAAAGGGATGTGCTGCGAGAAG</pe1>GCTAGA<cbc>[barcode1]</cbc><w1>CTCACTAACGAACACTGCGGAA</w1> -5'
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>-------->
</seq>
</pre>
<h3>(3) This is the product after the first extension:</h3>
<pre>
<seq>
3'- GCTACTGC<t7>ATTATGCTGAGTGATATCCC</t7>TATGGTGGTACC<pe1>GAGAAAGGGATGTGCTGCGAGAAG</pe1>GCTAGA<cbc>[barcode1]</cbc><w1>CTCACTAACGAACACTGCGGAA</w1> -5'
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1>
</seq>
</pre>
<h3>(4) Pooling, denature by NaOH, and get rid of top strand:</h3>
<pre>
<seq>
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1>
</seq>
</pre>
<h3>(5) Split again into wells in Barcode 2 plates, and primer extension:</h3>
<pre>
<align class="long">
<--------<w1>CTCACTAACGAACACTGCGGAA</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>A<sub>19</sub>B -5'
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1>---------->
</align>
</pre>
<h3>(6) This is the product of second extension:</h3>
<pre>
<align class="long">
3'- GCTACTGC<t7>ATTATGCTGAGTGATATCCC</t7>TATGGTGGTACC<pe1>GAGAAAGGGATGTGCTGCGAGAAG</pe1>GCTAGA<cbc>[barcode1]</cbc><w1>CTCACTAACGAACACTGCGGAA</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>A<sub>19</sub>B -5'
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>T<sub>19</sub>V -3'
</align>
</pre>
<h3>(7) Pool aggain, denautre by NaOH, get rid of top strand, neutralise, wash and ready to use (384 x 384 different combination of barcodes 1 & 2):</h3>
<pre>
<align class="long">
|--5'-/acrydite/iSpPC/ CGATGACG<t7>TAATACGACTCACTATAGGG</t7>ATACCACCATGG<pe1>CTCTTTCCCTACACGACGCTCTTC</pe1>CGATCT<cbc>[barcode1]</cbc><w1>GAGTGATTGCTTGTGACGCCTT</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>T<sub>19</sub>V -3'
</align>
</pre>
<br></br>
<h2>Step-by-step library generation (the 5'-/acrydite/iSpPC/ is omitted for simplicity)</h2>
<h3>(1) Anneal oligo-dT19V to mRNA and reverse transcription using MMLV inside droplets:</h3>
<pre>
<align class="long">
5'- XXXXXXXXXXXXXXXXXXXB(A)<sub>n</sub>
<-----V(T)<sub>19</sub><umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>[barcode1]</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>GGTACCACCATA<t7>GGGATATCACTCAGCATAAT</t7>GCAGTAGC -5'--|
</align>
</pre>
<h3>(2) RNaseH and DNA Pol I based second strand synthesis:</h3>
<pre>
<align class="long">
5'- XXX...XXXB(pA)<umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>[barcode1]</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>CCATGGTGGTAT<t7>CCCTATAGTGAGTCGTATTA</t7>CGTCATCG
XXX...XXXV(dT)<umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>[barcode1]</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>GGTACCACCATA<t7>GGGATATCACTCAGCATAAT</t7>GCAGTAGC -5'--|
↵
IVT starts from here
</align>
</pre>
<h3>(3) Break droplets and T7 <i>in vitro</i> transcription to amplify cDNA (resulting in single stranded RNA):</h3>
<pre>
<seq>
5'- <t7>G</t7>AUACCACCAUGG<pe1>CUCUUUCCCUACACGACGCUCUUC</pe1>CGAUCU<cbc>[barcode1]</cbc><w1>GAGUGAUUGCUUGUGACGCCUU</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>(dU)VXXX...XXX -3'
</seq>
</pre>
<h3>(4) Reverse transcription on amplified RNA (aRNA) using random priming (PE2-N6)</h3>
<pre>
<align class="long">
5'- <t7>G</t7>AUACCACCAUGG<pe1>CUCUUUCCCUACACGACGCUCUUC</pe1>CGAUCU<cbc>[barcode1]</cbc><w1>GAGUGAUUGCUUGUGACGCCUU</w1><cbc>[8-bp barcode2]</cbc><umi>[6-bp UMI]</umi>(dU)VXXX...XXX -3'
<-------NNNNNN
<pe2>TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCT</pe2> -5'
</align>
</pre>
<h3>(5) Fist strand cDNA of aRNA looks like this:</h3>
<pre>
<align class="long">
5'- <pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>XXX...XXXV(pA)<umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>[barcode1]</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>CCATGGTGGTAT<t7>C</t7> -3'
</align>
</pre>
<h3>(6) Adding PE1 PCR and PE2 PCR primers to amplify the first strand cDNA:</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GGTC<pe2>TCGGCATTCCTGCTGAAC</pe2>---------->
5'- <pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>XXX...XXXV(pA)<umi>[6-bp UMI]</umi><cbc>[8-bp barcode2]</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>[barcode1]</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>CCATGGTGGTAT<t7>C</t7> -3'
<----------<pe1>AGCACATCCCTTTCTC</pe1>
[6-bp sample index]<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(9) Final library structure:</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GGTC<pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>NN....NN</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>NNNNNN<p7>ATCTCGTATGCCGTCTTCTGCTTG</p7>
<p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(dT)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
<p5>Illumina P5</p5> <pe2>PE2</pe2> cDNA <umi>6bp</umi> <cbc>8bp</cbc> <w1>W1</w1> <cbc>barcode1</cbc> <pe1>PE1</pe1> 6bp <p7>Illumina P7</p7>
<umi>UMI</umi> <cbc>barcode2</cbc> sample index
</align>
</pre>
<br></br>
<h2>V2 Library sequencing (three steps):</h2>
<h3>(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, these are the cDNA reads):</h3>
<pre>
<align class="long">
5'- <pe2>GGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>--------->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(2) Add Index sequencing primer to sequence sample index (bottom strand as template):</h3>
<pre>
<align class="long">
5'- AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>----->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(3) Cluster regeneration, and add read 2 sequencing primer to sequence read 2 (top strand as template, these are the cell barcodes and UMI reads, at least 51 cycles):</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GGTC<pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>NN....NN</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>NNNNNN<p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3'
<-------------------------------------------TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1> -5'
</align>
</pre>
<br></br>
<h2>V3 Library sequencing (four steps), this is just a guess based on the github page description and sample fastq files, not confirmed:</h2>
<h3>(1) Add read 1 sequencing primer to sequence the first read (bottom strand as template, these are the cDNA reads):</h3>
<pre>
<align class="long">
5'- <pe2>GGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>--------->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(2) Add reverse complement of W1 primer primer to sequence barcode1 (bottom strand as template, 6 cycles):</h3>
<pre>
<align class="long">
5'- <w1>AAGGCGTCACAAGCAATCACTC</w1>----->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(3) Add Index sequencing primer to sequence sample index (bottom strand as template, 6 cycles)::</h3>
<pre>
<align class="long">
5'- AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>----->
3'- <p5>TTACTATGCCGCTGGTGGCTCTAGATGTG</p5>CCAG<pe2>AGCCGTAAGGACGACTTGGCGAGAAGGCTAGA</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>TTCCGCAGTGTTCGTTAGTGAG</w1><cbc>NN....NN</cbc>TCTAGC<pe1>CTTCTCGCAGCACATCCCTTTCTC</pe1>NNNNNN<p7>TAGAGCATACGGCAGAAGACGAAC</p7> -5'
</align>
</pre>
<h3>(4) Cluster regeneration, and add W1 primer to sequence barcode2 and UMI (top strand as template, seems to be 21 cycles, contain a bit of dT at the end):</h3>
<pre>
<align class="long">
5'- <p5>AATGATACGGCGACCACCGAGATCTACAC</p5>GGTC<pe2>TCGGCATTCCTGCTGAACCGCTCTTCCGATCT</pe2>XXX...XXXV(pA)<umi>NNNNNN</umi><cbc>NNNNNNNN</cbc><w1>AAGGCGTCACAAGCAATCACTC</w1><cbc>NN....NN</cbc>AGATCG<pe1>GAAGAGCGTCGTGTAGGGAAAGAG</pe1>NNNNNN<p7>ATCTCGTATGCCGTCTTCTGCTTG</p7> -3'
<--------------------<w1>TTCCGCAGTGTTCGTTAGTGAG</w1> -5'
</align>
</pre>
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