CTR:Notebook/PIRE/2014/04/24

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> PIRE RAD Library Preps - Troubleshooting</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==RAD Library Prep - Olive Sunbirds Plate 1==
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==Overview==
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* 96 well plate of 50ng of DNA 10uL
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After sequencing the first plate of RAD libraries, it appeared as though the P1 adapter and barcodes were missing. The hypothesis is that the P1 adapters failed to ligate and instead the P2 adapter ligated to both ends. When trying to selectively amplify the fragments with the P1 adapters, we actually amplified fragments with two P2 adapters.  This could happen because the P2 adapter contains binding sites for both the P1 adapter primer (underlined) and the P2 adapter primer (bold).
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* Digestion (2014-04-30)
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P1 adapter primer:
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**Mix and add to each well:
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5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA<b>CGCTCTTCCGATCT</b> 3'
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***0.68 uL water
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***1.20 uL NEBuffer 4
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***0.12 uL SbfI-HF
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**Incubation:
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***37 degrees for 60 minutes
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***65 degrees for 20 minutes
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* P1 adapter ligation (2014-04-30)
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P2 adapter primer:
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**Add 2 uL indexed P1 adapter (10nM) to each well
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5' <u>CAAGCAGAAGACGGCATACGA</u> 3'
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**Mix and add to each well:
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***1.28 uL water
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***0.40 uL NEBuffer 4
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***0.16 uL ATP
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***0.16 uL T4 Ligase
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**Incubation:
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*** 20 degrees for 60 minutes
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*** 65 degrees for 20 minutes
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* Clean up (2014-04-30)
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P2 adapter:
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**Take 5 uL from each well and combine to 1.7 mL tube
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5' <u>CAAGCAGAAGACGGCATACGA</u>GATCGGTCTCGGCATTCCTGCTGAAC<b>CGCTCTTCCGATC*T</b> 3'
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**AMPure bead clean up:
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***Use 480 uL beads to DNA (1:1)
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***2 washes of 800 uL 80% EtOH
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***Elute in 100 uL LowTE
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* Sonication (2014-05-01)
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Normally this would not happen because the match between the P1 adapter and the P1 primer is much better than the match between the P2 adapter and the P1 primer.  
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**BioRuptor NGS: 8 cycles of 15 seconds on, 90 seconds off, High Power
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[[Image:Sunbird1 sheared 2014-05-01.JPG|200px]]
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1) 1uL pre-sheared DNA,
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2) 2uL 100bp low scale ladder,
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3) 3uL sheared DNA
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*Blunt End Repair (2014-05-01)
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To test if this is in fact what happened, we first ordered a new, more specific P1 adapter primer that should not bind to the P2 adapter:
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**Mix:
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***50.0 uL fragmented DNA
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***5.5 uL water
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***3.0 uL End Prep Enzyme Mix
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***6.5 uL End Repair Reaction Buffer
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**Incubation:
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***20 degrees for 30 minutes
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***65 degrees for 30 minutes
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*P2 adapter ligation (2014-05-01)
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5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC*G 3'
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**Add to mix:
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***15.0 uL Blunt/TA Ligase Master Mix
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***2.5 uL P2 RAD adapter (5 uM)
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***1.0 uL Ligation Enhancer
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**Incubation:
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***20 degrees for 15 minutes
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*Size selection (2014-05-01)
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We then performed the following tests to 1) verify that the P2 adapter ligated to both ends and 2) verify that the old P1 adapter did not ligate.
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**Add 16.5 uL water for a total of 100 uL
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**AMPure bead size selection:
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***45 uL beads to remove large fragments
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***25 uL beads to remove small fragments
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***3 washes of 200 uL 80% EtOH
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***Elute in 20 uL LowTE
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*PCR amplification (2014-05-01)
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==Verification of P2 adapter ligation to both ends==
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**Mix:
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To see if the P2 adapter could have ligated to both ends, we start the RAD protocol from the sonication step, skipping the P1 adapter ligation step. If the end product amplifies with the old P1 primer, then only the P2 adapter could have ligated.
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***5 uL DNA
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***25 uL NEBNext High Fidelity 2x PCR Master Mix
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***18 uL water
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*** 1 uL P1 adapter primer (25 uM)
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*** 1 uL P2 adapter primer (25 uM)
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**PCR cycle:
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***98 degrees for 30 seconds
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***15 cycles of:
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****98 degrees for 10 seconds
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****65 degrees for 30 seconds
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****72 degrees for 30 seconds
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***72 degrees for 5 minutes
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[[Image:Sunbirds1 PCR 2014-05-01.JPG|200px]]
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1) 1 uL DNA template, 2) 2 uL 100 bp low scale ladder, 3) 5 uL PCR product
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*Bead clean up (2014-05-02)
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# Start with approximately 1700 ng DNA and sonicate in Bioruptor for 8 cycles of 15 seconds on, 90 seconds off (high power).
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**Use 45 uL AMPure beads (1:1)
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# Transfer 50 μL of sheared DNA to new PCR tube and add 5.5 μL water.
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**2 washes of 200 uL 80% EtOH
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# Perform steps for blunt end repair, adapter ligation, size selection, and PCR following the RAD protocol. The old P1 adapter primer is used in the PCR step, not the new one.
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**Elute in 30 uL LowTE
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# Run a gel using 1 μL of unamplified library template and 5 μL of PCR product.
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*Quant DNA library using PicoGreen and run (<3 ng/uL) on BioAnalyzer DNA High Sensitivity Chip
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[[Image:Testpcrcomparison.JPG|300px]]
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[[Image:Sunbirds1 bioanalyzer.jpg|450px]]
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*Dilute library to 10 nM in 10 uL and send to Berkeley for sequencing (Illumina HiSeq 100 SR) (2014-05-05)
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The PCR product is brighter than the template, suggesting successful amplification. Therefore, we can conclude that the P2 adapter did indeed ligate to both ends and was successfully amplified by the old P1 primer.
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==RAD Library Prep - ==
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==Verification of absence of P1 adapter==
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*
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We use leftover DNA library template from the failed first plate and re-do the final PCR step using the new P1 primer. Because it is more specific to the P1 adapter, the primer should not amplify segments with P2 adapters ligated to both ends. We expect to see amplification only if the P1 adapter successfully ligated.
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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[[Image:TestP1pcrcomparison.JPG|300px]]
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The first lane contains 1 μL of template and the second lane contains 5 μL of PCR product. Because there is no apparent amplification in the PCR product, we conclude that the P1 adapter did not ligate.
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__NOTOC__
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Current revision

PIRE RAD Library Preps - Troubleshooting Main project page
Next entry

Overview

After sequencing the first plate of RAD libraries, it appeared as though the P1 adapter and barcodes were missing. The hypothesis is that the P1 adapters failed to ligate and instead the P2 adapter ligated to both ends. When trying to selectively amplify the fragments with the P1 adapters, we actually amplified fragments with two P2 adapters. This could happen because the P2 adapter contains binding sites for both the P1 adapter primer (underlined) and the P2 adapter primer (bold).

P1 adapter primer: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3'

P2 adapter primer: 5' CAAGCAGAAGACGGCATACGA 3'

P2 adapter: 5' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T 3'

Normally this would not happen because the match between the P1 adapter and the P1 primer is much better than the match between the P2 adapter and the P1 primer.

To test if this is in fact what happened, we first ordered a new, more specific P1 adapter primer that should not bind to the P2 adapter:

5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC*G 3'

We then performed the following tests to 1) verify that the P2 adapter ligated to both ends and 2) verify that the old P1 adapter did not ligate.

Verification of P2 adapter ligation to both ends

To see if the P2 adapter could have ligated to both ends, we start the RAD protocol from the sonication step, skipping the P1 adapter ligation step. If the end product amplifies with the old P1 primer, then only the P2 adapter could have ligated.

  1. Start with approximately 1700 ng DNA and sonicate in Bioruptor for 8 cycles of 15 seconds on, 90 seconds off (high power).
  2. Transfer 50 μL of sheared DNA to new PCR tube and add 5.5 μL water.
  3. Perform steps for blunt end repair, adapter ligation, size selection, and PCR following the RAD protocol. The old P1 adapter primer is used in the PCR step, not the new one.
  4. Run a gel using 1 μL of unamplified library template and 5 μL of PCR product.

The PCR product is brighter than the template, suggesting successful amplification. Therefore, we can conclude that the P2 adapter did indeed ligate to both ends and was successfully amplified by the old P1 primer.

Verification of absence of P1 adapter

We use leftover DNA library template from the failed first plate and re-do the final PCR step using the new P1 primer. Because it is more specific to the P1 adapter, the primer should not amplify segments with P2 adapters ligated to both ends. We expect to see amplification only if the P1 adapter successfully ligated.

The first lane contains 1 μL of template and the second lane contains 5 μL of PCR product. Because there is no apparent amplification in the PCR product, we conclude that the P1 adapter did not ligate.

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