Chris:LabNotes/FateMapping/Calendar/2016/2016-5-18: Difference between revisions

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=Design of Phi29 Controls (Update #4.5, Update to 2nd UMI Oligo)=
==Background==
*I noticed that the original Second UMI Oligo (shown below, from 2/26/2016) had a minor error in it that was missing an A to account for the extra T overhang after BsrGI digestion.
**The general structure of the second UMI oligo is as follows:
<pre style="color: red"> 5' <u>EcoRI recognition site</u>|-----AmpR6.3, w/ <u>8N UMI2</u> instead of 6N index-----|<u>RevComp(TGCGTATCCGTGCT)</u> 3' </pre>
**Below is the Second UMI Oligo:
<u>'''Second UMI Oligo'''</u>: 5' <u>GAATT</u>CAAGCAGAAGACGGCATACGAGAT<u>NNNNNNNN</u>GCTAGGAACGATGAGCCTCCAAC<u>AGCACGGATACGCA</u> 3' (74nt)
*The correction should account for the T leftover after BsrGI digestion.  The digested sequence is shown below:
GTACA|GAATTC|GCAGTGTT|AATGA...CCGAC|8N|--LP--|--MS--|--RP--|TGCGTATCCGTGCT<u>'''T'''</u>
BsrGI|EcoRI |BtsaI  |  AmpF6.4    |UMI1|                  |Filler        Leftover T after BsrGI digestion
*We also want to move around the UMI2 sequence to right after the filler+T sequence in order to read the UMI2 on read 1.  This change will allow us to add a traditional indexing sequence.  It will also allow us to call UMI2 more confidently because, based on the previous 5/12 sequencing results, the quality score for the indexing read was much lower than the end of read 1.
*Consequently, the general structure of the second UMI oligo as as follows:
<pre style="color: red"> 5' <u>EcoRI recognition site</u>|-----AmpR6.3, w/ <u>6N Index</u>-----|'''8N UMI2'''|<u>RevComp(TGCGTATCCGTGCT'''T''')</u> 3' </pre>
Index    Sequence
11        GGCTAC <- These are based off of the Zhang Lab custom indices seq (RC)
12        CTTGTA
13        CCATGA
*Below are the three different second UMI oligos (different indices for each):
<u>'''Second UMI Oligo Idx11'''</u>: 5' <u>GAATT</u>CAAGCAGAAGACGGCATACGAGAT<u>GGCTAC</u>GCTAGGAACGATGAGCCTCCAAC<u>'''NNNNNNNN'''AAGCACGGATACGCA</u> 3'
<u>'''Second UMI Oligo Idx12'''</u>: 5' <u>GAATT</u>CAAGCAGAAGACGGCATACGAGAT<u>CTTGTA</u>GCTAGGAACGATGAGCCTCCAAC<u>'''NNNNNNNN'''AAGCACGGATACGCA</u> 3'
<u>'''Second UMI Oligo Idx13'''</u>: 5' <u>GAATT</u>CAAGCAGAAGACGGCATACGAGAT<u>CCATGA</u>GCTAGGAACGATGAGCCTCCAAC<u>'''NNNNNNNN'''AAGCACGGATACGCA</u> 3'
=Phi29 Slippage Rate Experiment - Method Comparison=
=Phi29 Slippage Rate Experiment - Method Comparison=
==Background==
==Background==

Revision as of 03:08, 19 May 2016

Design of Phi29 Controls (Update #4.5, Update to 2nd UMI Oligo)

Background

  • I noticed that the original Second UMI Oligo (shown below, from 2/26/2016) had a minor error in it that was missing an A to account for the extra T overhang after BsrGI digestion.
    • The general structure of the second UMI oligo is as follows:
 5' <u>EcoRI recognition site</u>|-----AmpR6.3, w/ <u>8N UMI2</u> instead of 6N index-----|<u>RevComp(TGCGTATCCGTGCT)</u> 3' 
    • Below is the Second UMI Oligo:
Second UMI Oligo: 5' GAATTCAAGCAGAAGACGGCATACGAGATNNNNNNNNGCTAGGAACGATGAGCCTCCAACAGCACGGATACGCA 3' (74nt)
  • The correction should account for the T leftover after BsrGI digestion. The digested sequence is shown below:
GTACA|GAATTC|GCAGTGTT|AATGA...CCGAC|8N|--LP--|--MS--|--RP--|TGCGTATCCGTGCTT
BsrGI|EcoRI |BtsaI   |  AmpF6.4    |UMI1|                  |Filler        Leftover T after BsrGI digestion
  • We also want to move around the UMI2 sequence to right after the filler+T sequence in order to read the UMI2 on read 1. This change will allow us to add a traditional indexing sequence. It will also allow us to call UMI2 more confidently because, based on the previous 5/12 sequencing results, the quality score for the indexing read was much lower than the end of read 1.
  • Consequently, the general structure of the second UMI oligo as as follows:
 5' <u>EcoRI recognition site</u>|-----AmpR6.3, w/ <u>6N Index</u>-----|'''8N UMI2'''|<u>RevComp(TGCGTATCCGTGCT'''T''')</u> 3' 
Index     Sequence
11        GGCTAC <- These are based off of the Zhang Lab custom indices seq (RC)
12        CTTGTA
13        CCATGA
  • Below are the three different second UMI oligos (different indices for each):
Second UMI Oligo Idx11: 5' GAATTCAAGCAGAAGACGGCATACGAGATGGCTACGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3'
Second UMI Oligo Idx12: 5' GAATTCAAGCAGAAGACGGCATACGAGATCTTGTAGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3'
Second UMI Oligo Idx13: 5' GAATTCAAGCAGAAGACGGCATACGAGATCCATGAGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3'

Phi29 Slippage Rate Experiment - Method Comparison

Background

Phi29 Error "PCR" Error Sequencing Error
PCR-based 0.085 0.277 3.36e-7
Culture-based 0.369 2.77e-5 4.86e-6
  • All of this analysis can be found on genemapster in </media/6TB_slot3/cjwei/Fate_Mapping/Non-Capture_Analysis/seq_error_[culture/PCR]> and is summarized in my 5/18 lab meeting presentation
    • The above calculates suggest that, while the culture-based method has a very low "PCR" error rate (as expected because bacteria should replicate inserts with very high fidelity), it also exhibited much higher Phi29 error.
  • We're not sure exactly what caused these vastly different error rates for phi29 slippage, so we'll be running a bunch of different tests this week to figure out what's going on.

Procedure

RCA

                       1x     3.5x
Template (1/10d)       5ul    -- <- used samples 1-3 (2nt, 3nt, 4nt)
RCA Primer (100uM)     2.5    8.75
dNTP                   0.8    2.8
10x Buff               2      7
Phi29                  1      3.5
Water                  8.7    30.45
-----------------------------------
                       20ul
  • Thermocycler: 37C 3hr -> 65C 10min -> 10C hold
  • I then performed ETOH precipitation and eluted into 20ul. These RCA reactions will then be aliquoted and used for various tests in order to keep most things consistent.

Test 1: Culture-based Method

BsrGI Digestion (2x, one for Culture and another for PCR. Mix eluent together before continuing to next step)

  • I'll be using only 1/5 of the purified RCA product for this test. Consequently, I used the following mastermix for each of the three reactions/samples:
                         1x
RCA product              4ul
10x Cutsmart             5
100uM RE_BsrGI Oligo     5 <- I used the Redesigned Oligo from 4/15
Water                    25
-------------------------------
                         39ul
  • Thermocycler: 94C 2min -> 37C 3min -> Add 1ul BsrGI-HF -> 37C 2hr -> 80C 20min
  • I then purified this digestion product using ETOH purification and eluted into 22ul and saved 2ul of that for gel quantification

Second Strand Synthesis (2x, one for Culture and another for PCR. Mix eluent together before continuing to next step)

  • After BsrGI digestion (and ETOH precipitation, eluted in 20ul water), we did second strand synthesis with the addition of the AmpR6.3 sequence (along with an 8N UMI2 replacing the index).
  • The following is the mastermix fo rthe second strand synthesis reaction:
                  1x
2nd UMI Oligo     5ul <- I used v4 of the 2nd UMI Oligo from 2/26/2016
KAPA HiFi         15
Template          10
  • We did two reactions per sample, using up all of the BsrGI product. After second strand synthesis, we combined both reactions, did ETOH precipitation, and eluted in 15ul.
  • At this point, we have 30ul of second strand synthesis product per reaction because we did 2x for BsrGI digestion and Second Strand Synthesis. Use 15ul to continue to de-phosphorylation and use 15ul to use for PCR in Test 2 below

De-phosphorylation with rSAP

  • After second strand synthesis, we must remove the phosphate group on the 5' end, which was left after BsrGI digestion. The leftover 5' phosphate group would prevent the blunt-end PCR product from ligating into the Zero Blunt TOPO plasmid.
  • Below is the mastermix for the rSAP dephosphorylation:
                 1x
Template         15ul
10x Cutsmart     2
rSAP             1
Water            2
  • Thermocycler: 37C 30min -> 65C 5min -> 10C forever.
  • We will do size selection of the second strand synthesis product and perform ETOH precipitation and elute in 6ul water (4ul is added to the TOPO reaction)

TOPO Blunt-end Ligation and Transformation

  • We will be doing four transformations (three samples + 1 positive control using the previous PCR of the given control insert)
rxn     Sample     Water     Salt     TOPO
1 (2nt) 4ul        0ul       1ul      1ul
2 (3nt) 4          0         1        1
3 (4nt) 4          0         1        1
4       (1/10d) 1  3         1        1 <- By using a 1/10d of the control insert, we can mimic 1ng of input into the TOPO reaction
Add DNA (10pg-100ng) and mix gently (do not pipette up/down)
                           |
                           V
                       30min Ice
                           |
                           V
                       42C 30sec
                           |
                           V
                       2min Ice <- I wasn't as stringent before on this waiting step.  So will make sure to wait 2min prior to adding SOC in order to allow bacteria to take up plasmids
                           |
                           V
    Add 250ul SOC and shake @37C for 3hr at 300rpm
                           |
                           V
                 Spread onto plates

Wash LB Agar+Kanamycin Plates

  • I washed the plates using the same procedure as outlined in <http://www.boneandcancer.org/protocolsa13.htm>
  • In summary, I added 4ml LB+Kanamycin broth to each plate and allowed solution to sit for a few minutes. Afterwards, I shook the plates in order to release the colonies into solution.
    • I tried pipetting directly on some colonies that were stuck in order to release.
    • Some agar was transferred into the solution because sometimes the p1000 pipette would accidentally scrape the gel.
  • I then proceeded with miniprep as per instructions with only 3 tubes (combined all plates belonging to the same reaction together)

EcoRI/BtsaI Double Digestion

                 rxn1     rxn2     rxn3
EcoRI-HF         1ul      1        1
DNA              20       39       40
10x Cutsmart     5        5        5
Water            29       10       9
  • Incubate: 37C 2hrs -> Add 1ul BtsaI (remove 2.5ul to run gel checking EcoRI digestion) -> 55C Overnight -> 65C 20min (deactivate EcoRI) -> 10C forever

Test 2: PCR-based Method

P5/P7 Library PCR

  • We want to take the same exact BsrGI+Second Strand Synthesis product as above (take 15ul of the above second strand synthesis reaction prior to rSAP)
  • Instead of continuing to ligation and transformation, we want to use PCR to amplify our library instead. To do this, we want to design new primers matching the already attached P5/P7 regions of the second strand product. This is the same sequence as used in the Illumina primer premix in the KAPA library quantification kit (File:Cw 20160518 KAPA Library Quantification Illumina TDS2.pdf):
P5 Primer: 5' AATGATACGGCGACCACCGA 3'
P7 Primer: 5' CAAGCAGAAGACGGCATACGA 3'
  • Mastermix recipe:
                  Init Conc     1x       3.5x
Kapa HiFi Mix     2x            15ul     52.5ul
P5 Primer         10uM          2.5      8.75
P7 Primer         10uM          2.5      8.75
Template          --            10       -- (Add Individually)
------------------------------------------------
                                30ul
  • Thermocycler: (we use the same thermocycler profile as we usually use for probe capture but make a few adjustments to match suggested KAPA HiFi denaturing temperatures)
95C 3min
(95C 10sec -> 58C 30sec -> 72C 20sec)x8
(95C 10sec -> 72C 20sec)x12 cycles
72C 3min
Hold 15C
  • Use all 30ul of the product in order to do size selection and move forward with sequencing (PE150 + 8N Index)

Test 3: phi29 Second Strand Synthesis, Ligation, and Direct Sequencing

Test 4: Sanger Sequencing Approach