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

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*Thermocycler: 37C '''3hr''' -> 65C 10min -> 10C hold
*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.
*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.
*Below are the concentrations after RCA+ETOH precipitation:
rxn    Conc (ng/ul)    Total (20ul, ug)
1      141              2.82
2      139              2.78
3      136              2.72
==Test 1: Culture-based Method==
==Test 1: Culture-based Method==
===BsrGI Digestion (<span style="background:yellow"><u>2x, one for Culture and another for PCR.  Mix eluent together before continuing to next step</u></span>)===
===BsrGI Digestion (<span style="background:yellow"><u>2x, one for Culture and another for PCR.  Mix eluent together before continuing to next step</u></span>)===
Line 85: Line 91:
  10x Cutsmart            5
  10x Cutsmart            5
  100uM RE_BsrGI Oligo    5 <- I used the '''Redesigned''' Oligo from 4/15
  100uM RE_BsrGI Oligo    5 <- I used the '''Redesigned''' Oligo from 4/15
  Water                    25
  Water                    35
  -------------------------------
  -------------------------------
                           39ul
                           49ul
*Thermocycler: 94C 2min -> 37C 3min -> Add 1ul BsrGI-HF -> 37C 2hr -> 80C 20min
*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
*I then purified this digestion product using ETOH purification and eluted into 22ul and saved 2ul of that for gel quantification
===Second Strand Synthesis (<span style="background:yellow"><u>2x, one for Culture and another for PCR.  Mix eluent together before continuing to next step</u></span>)===
===Second Strand Synthesis (<span style="background:yellow"><u>2x, one for Culture and another for PCR.  Mix eluent together before continuing to next step</u></span>)===
*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).
*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:
*The following is the mastermix for the second strand synthesis reaction:
                   1x
                   1x
  2nd UMI Oligo    5ul <- I used v4.5 of 2nd UMI Oligo designed today (<span style="background:yellow">used '''Idx11''' for culture-based method, '''Idx12''' for PCR-based method</span>)
  2nd UMI Oligo    5ul <- I used v4.5 of 2nd UMI Oligo designed today (<span style="background:yellow">used '''Idx11''' for culture-based method, '''Idx12''' for PCR-based method</span>)
Line 99: Line 106:
*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.
*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.
*<span style="background:yellow"><u>'''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'''</u></span>
*<span style="background:yellow"><u>'''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'''</u></span>
===De-phosphorylation with rSAP===
===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.
*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.
Line 225: Line 233:
                                     20ul
                                     20ul
*Thermocycler: 16C Overnight -> 65C 10min -> 10C forever
*Thermocycler: 16C Overnight -> 65C 10min -> 10C forever
*Run size selection and ETOH purification to extract correct insert and elute in 10ul water
*Run size selection and ETOH purification to extract correct insert and elute in 20ul water


==Test 4: Sanger Sequencing Approach==
==Test 4: Sanger Sequencing Approach==
*We want to determine the phi29 error independent of any other factor (i.e. independent of Illumina sequencing error).  To do this, we'll be submitting some of the "Ligate Direct Seq 2nd UMI Oligo" product to Sanger sequencing.
*Based on the chemistry of Sanger sequencing, we should only be obtaining the original phi29 replicated sequence instead of the 2nd strand synthesis product.  This is because we'll be using the P7 primer as the sequencing primer submitted to Genewiz.  This primer is consequently strand specific and sufficiently downstream of the microsatellite region to account for the low quality bases at the beginning of Sanger sequencing runs.
*To submit the product to Genewiz, we need to submit a pre-mix of the sample + P7 primer according to the specific concentrations:
DNA Type                  DNA Length    Template Conc    Total Template    Primer pmol    Pre-mix Volume
Purified PCR Products    <500bp        ~1ng/ul          ~10ng              25pmol          15ul
*Consequently, we want to add:
Ligation product    x ul <- We want ~10ng product
10uM P7 Primer      2.5  <-(same as Test #2 above)
Water                15-x
---------------------------
                      15ul
*Submit to Genewiz as pre-mix and analyze trace file

Latest revision as of 21:33, 3 June 2016

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

Update 2nd UMI Oligo[edit]

  • 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 original 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 original 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 updated 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):
2nd UMI Oligo Idx11: 5' GAATTCAAGCAGAAGACGGCATACGAGATGGCTACGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3' (81nt)
2nd UMI Oligo Idx12: 5' GAATTCAAGCAGAAGACGGCATACGAGATCTTGTAGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3' (81nt)
2nd UMI Oligo Idx13: 5' GAATTCAAGCAGAAGACGGCATACGAGATCCATGAGCTAGGAACGATGAGCCTCCAACNNNNNNNNAAGCACGGATACGCA 3' (81nt)

Direct Seq 2nd UMI[edit]

  • In order to further validate the protocol, we want to be able to do direct sequencing without any need to amplify over the microsatellite region with another enzyme or use culture methods. Instead, we want to perform second strand synthesis directly on the RCA product, linearize, and then add the correct sequencing adapters (AmpR6.3). Below is a description of the method:
1) RCA - Using previously designed RCA primer.  Perform ETOH precipitation.
2) Prior to linearization, perform second strand synthesis using the redesigned RE_BsrGI primer and phi29. Perform ETOH precipitation.
3) Linearize by BsrGI digestion.  This leaves an overhang at the 5' end with (5' GTAC 3')
       BsrGI|EcoRI |BtsaI   |AmpF6.4      |UMI1|                  |Filler        |BsrGI
   5'  GTACA|GAATTC|GCAGTGTT|AATGA...CCGAC|8N|--LP--|--MS--|--RP--|TGCGTATCCGTGCTT       3'
   3'      T|CTTAAG|CGTCACAA|TTACT...GGCTG|8N'|-LP'-|--MS'-|--RP'-|ACGCATAGGCACGAACATG   5'
4) Remove 5' phosphate groups from the inserts by treating with rSAP.  This will prevent self ligation of inserts
5) Anneal "Direct Seq 2nd UMI" oligo and perform ligation with T4 DNA ligase, which will repair the nick formed by joining the 3' hydroxyl end of the BsrGI digested product and the 5' phosphate end of the "Direct Seq 2nd UMI"
6) Cut with BtsaI, resulting in one of the strands being a fully formed read
  • Consequently, the "Direct Seq 2nd UMI" oligo must contain the following sequences:
5' /5Phos/GTAC 3' - Complementary to the sticky end
NNNNNNNN - 8N UMI2
reverse comp(AmpR6.3), w/ 6N Index - AmpR6.3 sequence with 6N Index (Index 14, RC sequence is AAGACA, so we must use the RC(AAGACA) = TGTCTT)
  • The following is the "Direct Seq 2nd UMI" oligo sequence:
Direct Seq 2nd UMI: 5' /5Phos/GTACNNNNNNNNGTTGGAGGCTCATCGTTCCTAGCTGTCTTATCTCGTATGCCGTCTTCTGCTTG 3'

Phi29 Slippage Rate Experiment - Method Comparison[edit]

Background[edit]

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[edit]

RCA[edit]

                       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.
  • Below are the concentrations after RCA+ETOH precipitation:
rxn     Conc (ng/ul)     Total (20ul, ug)
1       141              2.82
2       139              2.78
3       136              2.72

Test 1: Culture-based Method[edit]

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

  • 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                    35
-------------------------------
                         49ul
  • 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)[edit]

  • 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 for the second strand synthesis reaction:
                  1x
2nd UMI Oligo     5ul <- I used v4.5 of 2nd UMI Oligo designed today (used Idx11 for culture-based method, Idx12 for PCR-based method)
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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

                 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
  • Perform size selection and perform ETOH purification, eluting in 10ul water for sequencing

Test 2: PCR-based Method[edit]

P5/P7 Library PCR[edit]

  • 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, perform ETOH purification and elute in 10ul water, and move forward with sequencing (PE150 + 8N Index)

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

  • In this test, we want to use phi29 instead of KAPA Hifi to do second strand synthesis. Consequently, we can still use the same RCA product as before, but we want to perform the following steps instead:
                          RCA
                           |
                           V
phi29 second strand synthesis (using redesigned RE_BsrGI oligo as primer)
Incubate for only 10min at 37C
                           |
                           V
           Restriction enzyme dig with BsrGI
   (add rSAP to digested inserts to prevent self-ligation)
                           |
                           V
    Ligate Direct Seq 2nd UMI Oligo with T4 DNA Ligase
                           |
                           V
     Sequence (one strand will have fully formed read)

phi29 Second Strand Synthesis[edit]

  • As mentioned above, we will use the same RCA product as used for the other tests, but this time, doing phi29 second strand synthesis using the following mastermix:
                         1x     3.5x
RCA Product              4ul    --
100uM RE_BsrGI Oligo     2.5    8.75<- I used the Redesigned Oligo from 4/15
dNTP                     0.8    2.8
10x Buff                 2      7
Phi29                    1      3.5
Water                    9.7    33.95
---------------------------------------
                         20ul
  • Thermocycler: 37C 10min -> 65C 10min -> 10C hold
  • I then performed ETOH precipitation and eluted into 10ul. All 10ul of second strand product will be used for BsrGI digestion.

BsrGI Digestion and rSAP De-Phosphorylation[edit]

  • I'll be using all of the phi29 second strand synthesis product from the above step to do BsrGI digestion as follows:
phi29 Second Strand Synth     10ul
10x Cutsmart                  5
Water                         34
BsrGI-HF                      1
-----------------------------------
                              50ul
  • Thermocycler: (NOTE: NO 94C DENATURING) 37C 2hr -> 80C 20min -> 10C forever -> Add 1ul rSAP -> 37C 1hr -> 65C 5min -> 10C forever
  • I then purified this digestion product using ETOH precipitation and eluted into 12ul and saved 2ul of that for gel quantification

Ligate Direct Seq 2nd UMI Oligo[edit]

  • We then want to ligate the Direct Seq 2nd UMI Oligo by taking advantage of the sticky end leftover from BsrGI digestion. To do this, we'll be using T4 DNA Ligase (NEB, 400,000U/ml)
  • Below is the reaction used for T4 DNA ligation:
                                   1x
10x T4 DNA Ligase Buff             2ul
BsrGI Dig Product                  10 <- Assuming that we get about 1ug from RCA product, the max BsrGI product is ~8pmol
10uM Direct Seq 2nd UMI Oligo      1 <- We want to use ~1:1 ratio of digested product:Direct Seq 2nd UMI Oligo, so only add ~8pmol to reaction
T4 DNA Ligase                      1
Water                              6
-----------------------------------------
                                   20ul
  • Thermocycler: 16C Overnight -> 65C 10min -> 10C forever
  • Run size selection and ETOH purification to extract correct insert and elute in 20ul water

Test 4: Sanger Sequencing Approach[edit]

  • We want to determine the phi29 error independent of any other factor (i.e. independent of Illumina sequencing error). To do this, we'll be submitting some of the "Ligate Direct Seq 2nd UMI Oligo" product to Sanger sequencing.
  • Based on the chemistry of Sanger sequencing, we should only be obtaining the original phi29 replicated sequence instead of the 2nd strand synthesis product. This is because we'll be using the P7 primer as the sequencing primer submitted to Genewiz. This primer is consequently strand specific and sufficiently downstream of the microsatellite region to account for the low quality bases at the beginning of Sanger sequencing runs.
  • To submit the product to Genewiz, we need to submit a pre-mix of the sample + P7 primer according to the specific concentrations:
DNA Type                  DNA Length     Template Conc     Total Template     Primer pmol     Pre-mix Volume
Purified PCR Products     <500bp         ~1ng/ul           ~10ng              25pmol          15ul
  • Consequently, we want to add:
Ligation product     x ul <- We want ~10ng product
10uM P7 Primer       2.5  <-(same as Test #2 above)
Water                15-x
---------------------------
                     15ul
  • Submit to Genewiz as pre-mix and analyze trace file