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= Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit = | = Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit = | ||
[[noi:DMR220k_LabNotes#2014|''' | [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] | ||
* REPEAT experiment of 2014-10-22 | * REPEAT experiment of 2014-10-22 | ||
* [[Media:KAPA_Hyper_Prep_Kit_TDS.pdf|'''KAPA Hyper Prep Kit Manual''']] | * [[Media:KAPA_Hyper_Prep_Kit_TDS.pdf|'''KAPA Hyper Prep Kit Manual''']] | ||
Line 15: | Line 15: | ||
#4 6P-3 1.57 | #4 6P-3 1.57 | ||
* Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. | * Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. | ||
* KAPA Hyper Prep reaction set up (exactly in the instruction: | * KAPA Hyper Prep reaction set up (exactly in the instruction): | ||
:- 50ul of fragmented DNA | :- 50ul of fragmented DNA | ||
:- 7ul of End repair & dA-tailing buffer | :- 7ul of End repair & dA-tailing buffer | ||
Line 53: | Line 53: | ||
| NTC||0.00||0.00||5.00||25.00||30.00 | | NTC||0.00||0.00||5.00||25.00||30.00 | ||
|} | |} | ||
:- Mix | :- Mix well | ||
:- 20C, 30min | :- 20C, 30min | ||
:- 65C, 30min, | :- 65C, 30min, | ||
Line 87: | Line 87: | ||
| Total||55.00 | | Total||55.00 | ||
|} | |} | ||
* I used 1:20 diluted Truseq adaptor prepared | * I used 1:20 diluted Truseq adaptor prepared in June 2014 | ||
<u>'''KAPA Hyper Prep indexing'''</u> | |||
:{| {{table}} border = 1 | :{| {{table}} border = 1 | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample IDs''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Sample IDs''' | ||
Line 107: | Line 108: | ||
:- 20C, 15min | :- 20C, 15min | ||
:- Continue to SPRI clean up immediately | :- Continue to SPRI clean up immediately | ||
* Final conc. of adaptor in 55ul reaction is ~ | * Final conc. of adaptor in 55ul reaction is ~13nM, which is very close to 14nM recommended by the kit. | ||
= NEXTflex Cell Free DNA Seq Kit = | |||
'''Sample list (the same as above)'''<br> | |||
'''No.''' '''Sample IDs''' '''Conc. (ng/ul)''' | |||
#1 NC-2 0.325 | |||
#2 NC-3 0.538 | |||
#3 6P-2 1.10 | |||
#4 6P-3 1.57 | |||
* Note: I also reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. | |||
* NEXTflex Cell Free DNA Seq reaction set up (exactly in the instruction): | |||
:- 32ul of cell-free DNA | |||
:- 15ul of NEXTflex End-Repair & Adenylation Buffer mix | |||
:- 3ul of NEXTflex End-Repair & Adenylation Enzyme mix | |||
:- Total reaction: 50ul | |||
== End repair & Adenylation == | |||
==== End repair & Adenylation reaction mix ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:220px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''5.2x rxn''' | |||
|- | |||
| End repair & Adenylation buffer mix||15.00||7.50||39.00 | |||
|- | |||
| End repair & Adenylation enzyme mix||3.00||1.50||7.80 | |||
|- | |||
| Total||18.00||9.00|| | |||
|} | |||
==== Reaction set up ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' | |||
|- | |||
| NC-2||0.33||3.08||9.00||12.92||25.00 | |||
|- | |||
| NC-3||0.54||1.86||9.00||14.14||25.00 | |||
|- | |||
| 6P-2||1.10||0.91||9.00||15.09||25.00 | |||
|- | |||
| 6P-3||1.57||0.64||9.00||15.36||25.00 | |||
|- | |||
| NTC||0.00||0.00||9.00||16.00||25.00 | |||
|} | |||
:- Mix the reaction well | |||
:- 22C, 20min | |||
:- 72C, 20min, | |||
:- Hold 4C | |||
:- Proceed to Adaptor ligation immediately | |||
== Adaptor ligation == | |||
* For this kit, it suggest to dilute the 25uM adaptor 1:8 or 9X (2777.78nM) and use 2.5ul in total reaction 100ul. The final conc. of adaptor in ligation reaction is 69.4nM, which is ~5 time more adaptor concentration suggested by KAPA Hyper Prep kit. | |||
* Truseq adaptor concentration is 15uM. I diluted it to 2777.78nM by mixing 2ul of 15uM adaptor with 8.8ul H2O and use 1.25ul in total 50ul of ligation reaction. | |||
==== Adaptor ligation reaction set up ==== | |||
<u>'''NEXTflex indexing'''</u> | |||
:{| {{table}} border = 1 | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | |||
|- | |||
| NC-2||6 | |||
|- | |||
| NC-3||7 | |||
|- | |||
| 6P-2||8 | |||
|- | |||
| 6P-3||10 | |||
|- | |||
| NTC||6 | |||
|} | |||
* The Enzyme mix is already in buffer, so I | |||
:- Add 1.25ul of 2777.7nM TruSeq adaptor | |||
:- Add 23.75ul of NEXTflex Ligase enzyme mix directly to dA-tailed DNA | |||
:- Mix well | |||
:- 22C, 15min | |||
:- Continue to SPRI clean up immediately | |||
==== <u>1X AMPure bead purification</u> ==== | ==== <u>1X AMPure bead purification</u> ==== | ||
* The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead | |||
** KAPA Hyper prep kit: does 1 round of 1X AMPure bead purification | |||
** NEXTflext: does 2 round of 1X AMPure bead purification. I guess due to the high amount of adaptor in ligation reaction (5X more than KAPA Hyper) | |||
- Aliquot AMPure bead in 1.5mL tube | |||
:- Add 55ul (for KAPA Hyper) or 50ul (for NEXTflex) AMPure bead. Mix by pipetting 10x | |||
:- Sit for 15min (NEXTflex suggested 5min) | |||
:- Transfer to sit on magnet for 5min | |||
:- Wash twice with 200ul freshly prepared 80% EtOH | |||
:- Dry the bead for 3-5min | |||
:- Resuspend with 21ul EB buffer | |||
::- For NEXTflex, I resuspended with 50ul EB and repeat 1X vol AMPure bead purification following the procedures above. | |||
:- Continue to bisulfite conversion immediately | |||
<br> | |||
== Bisulfite conversion == | |||
* I performed bisulfite conversion using the same procedure following manufacturer's instruction of EZ DNA Methylation-Lightning kit and elute with 32ul elution buffer. | |||
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br> | |||
:- Aliquot 6.12mL of binding buffer & mix with 10.2ul of 10ng/ul tRNA | |||
:- Aliquot 5.1mL of Washing buffer | |||
:- Aliquot 2.1mL of Desulfonation buffer | |||
:- Aliquot 340ul of Elution buffer | |||
== Amplification == | |||
* Since from the previous experiment, I saw very low amplification by doing 1 round of PCR at high cycle number. I would follow the old optimize protocol by doing 2 round PCR. the amplification was lower than the WGBS using Thermoscientific system I did before. I would increase 1 round PCR to 15 cycle instead of 12cycles. Do 1X AMPure bead purification and continue to 2nd round PCR. | |||
* I use amplification primers in our lab designed for TruSeq adaptor. | |||
==== 1st round PCR ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''11.5x rxn mix''' | |||
|- | |||
| Bis-cvt DNA||||||||||30.00||0.00 | |||
|- | |||
| 10X Reaction buffer||10||X||1||X||5.00||57.50 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||1.25||14.38 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||1.50||17.25 | |||
|- | |||
| 50X SYBG||50||X||0.4||X||0.40||4.60 | |||
|- | |||
| PfuTurbo Cx||2.5||Unit/ul||2.5||unit||1.00||11.50 | |||
|- | |||
| H2O||||||||||10.85||124.78 | |||
|- | |||
| Total ||||||||||50.00|| | |||
|} | |||
:- Aliquot 20 + 30ul BIS-DNA template | |||
:- Mix well | |||
::''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 15 --> 72C for 2min'' | |||
:- Took 3ul for PAGE verification with no bead purify | |||
[[File:ZhangLab_2 2014-10-24 22hr 44min_1st-PCR-KAPA-NEXTflex-3ulPCR.jpg| 500px]] | |||
:- Purify with 1X AMPure beads | |||
:- Elute with 40ul EB Buffer | |||
:- Continue to 2nd round PCR. No PAGE verification. | |||
==== 2nd round PCR (QUICK TEST to monitor PCR cycle number) ==== | |||
* I did a quick test in small volume by using 2ul of bead purified 1st round PCR in total 10ul. Then split into 2X of 4.5ul and stopped based on qPCR curve. | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''12 rxn mix''' | |||
|- | |||
| Purified 1st round DNA||||||||||2.00||0.00 | |||
|- | |||
| 5X Phusion HF buffer||5||X||1||X||2.00||24.00 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||0.25||3.00 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||0.30||3.60 | |||
|- | |||
| 50X SYBR||50||X||0.4||X||0.08||0.96 | |||
|- | |||
| Phusion HF||2||unit/ul||||||0.10||1.20 | |||
|- | |||
| H2O||||||||||5.27||63.24 | |||
|- | |||
| Total||||||||||10.00|| | |||
|} | |||
:- Aliquot 8 + 2ul DNA template | |||
:- Mix well. | |||
:- Split 4.5ul | |||
::''98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5, 6, and 7 cycles --> 72C for 2min'' | |||
'''KAPA Hyper prep''' | |||
:- Sample NC2 & NC3 stopped at 5X and 7X | |||
:- Sample 6P2 & 6P3 stopped at 5X and 6X | |||
'''NEXTflex''' | |||
:- Sample NC2 & NC3 stopped at 5X and 7X | |||
:- Sample 6P2 & 6P3 stopped at 5X and 7X | |||
:- Verify all PCR product in 6% TBE gel | |||
[[File:ZhangLab_2 2014-10-27 12hr 50min_2ndPCR-KAPA-NEXTflex-5x.jpg| 500px]] | |||
[[File:ZhangLab_2 2014-10-27 12hr 50min_2ndPCR-KAPA-NEXTflex-6x-7x.jpg| 500px]] | |||
* From the gel image, XXXXX | |||
:- I do 2nd round PCR in the large volume by using all 1st round PCR as template. I reduce template amount of 6P samples by KAPA Hyper Prep kit to 10% and increase 2nd round PCR to 6 cycles. For the rest samples, I use 20% of DNA template from 1st round PCR and ran 2nd round PCR for 8 cycles. | |||
==== 2nd round PCR (use all 1st round PCR) ==== | |||
====<u> Mix 1: Low signal</u> ==== | |||
* Sample: KAPA Hyper Prep, NC2 & NC3 all sample of NEXTflex | |||
{| {{table}} border = 1 | |||
| align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''6.2 rxn mix''' | |||
|- | |||
| Purified 1st round DNA||||||||||36.00||0.00 | |||
|- | |||
| 5X Phusion HF buffer||5||X||1||X||36.00||223.20 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||4.50||27.90 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||5.40||33.48 | |||
|- | |||
| 50X SYBR||50||X||0.4||X||1.44||8.93 | |||
|- | |||
| Phusion HF||2||unit/ul||||||1.80||11.16 | |||
|- | |||
| H2O||||||||||94.86||588.13 | |||
|- | |||
| Total||||||||||180.00|| | |||
|} | |||
:- Aliquot 144ul + 36ul of template | |||
:- Split 50ul to each well of 8-tube strip | |||
::''98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X '''8cycles''' --> 72C for 2min'' | |||
====<u> Mix 2: High signal</u> ==== | |||
* Sample: KAPA Hyper Prep 6P2 & 6P3 | |||
{| {{table}} border = 1 | |||
| align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''2.2 rxn mix''' | |||
|- | |||
| Purified 1st round DNA||||||||||36.00||0.00 | |||
|- | |||
| 5X Phusion HF buffer||5||X||1||X||72.00||158.40 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||9.00||19.80 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||10.80||23.76 | |||
|- | |||
| 50X SYBR||50||X||0.4||X||2.88||6.34 | |||
|- | |||
| Phusion HF||2||unit/ul||||||3.60||7.92 | |||
|- | |||
| H2O||||||||||225.72||496.58 | |||
|- | |||
| Total||||||||||360.00|| | |||
|} | |||
:- Aliquot 324ul + 36ul of template | |||
:- Split 50ul to each well of 8-tube strip | |||
::''98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X '''6 cycles''' --> 72C for 2min'' | |||
:- Do 1 round (before I do 2 rounds, but this experiment the adaptor dimer was much fainter than before) bead purification of all PCR product with 1X AMPure beads and elute with 50ul EB buffer. | |||
:- Verified 2ul of bead purified sequencing library in 6% TBE gel | |||
[[File:ZhangLab_2 2014-10-27 17hr 01min_2ul_PAGEverify_KAPAHyper_NEXTflex_WGBS.jpg|500px]] | |||
:- Took 1.5ul for Qubit dsDNA HS quantification | |||
==== Qubit dsDNA HS quantification result ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 46ul''' | |||
|- | |||
| NC-2_KAPA||6.92||ng/mL||1.5||133.33||0.92||42.44 | |||
|- | |||
| NC-2_KAPA||23.2||ng/mL||1.5||133.33||3.09||142.29 | |||
|- | |||
| 6P2_KAPA||137||ng/mL||1.5||133.33||18.27||840.27 | |||
|- | |||
| 6P3_KAPA||72.6||ng/mL||1.5||133.33||9.68||445.28 | |||
|- | |||
| NC2_NEXTflex||3.86||ng/mL||1.5||133.33||0.51||23.67 | |||
|- | |||
| NC3_NEXTflex||8.29||ng/mL||1.5||133.33||1.11||50.85 | |||
|- | |||
| 6P2_NEXTflex||80.5||ng/mL||1.5||133.33||10.73||493.73 | |||
|- | |||
| 6P3_NEXTflex||25.2||ng/mL||1.5||133.33||3.36||154.56 | |||
|} | |||
<br> | |||
=== Comparison of the PCR cycle number and yield of these samples to the previous protocol using only Klenow fragment, exo- === | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''1st PCR''' | |||
| align="center" style="background:#f0f0f0;"|'''2nd PCR''' | |||
| align="center" style="background:#f0f0f0;"|'''Total PCR cycles''' | |||
| align="center" style="background:#f0f0f0;"|'''Yield (ng)''' | |||
|- | |||
| <span style="color:crimson">'''Klenow exo-'''<span style="color:crimson">|||||||| | |||
|- | |||
| NC2||12||7||19||111.20 | |||
|- | |||
| NC3||12||7||19||144.00 | |||
|- | |||
| 6P2||12||6||18||756.00 | |||
|- | |||
| 6P3||12||6||18||369.60 | |||
|- | |||
| <span style="color:crimson">'''KAPA Hyper Prep kit'''<span style="color:crimson">|||||||| | |||
|- | |||
| NC2||15||8||23||42.44 | |||
|- | |||
| NC3||15||8||23||142.29 | |||
|- | |||
| 6P2||15||6||21||840.27 | |||
|- | |||
| 6P3||15||6||21||445.28 | |||
|- | |||
| <span style="color:crimson">'''NEXTflex Cell Free DNA-Seq kit'''<span style="color:crimson">|||||||| | |||
|- | |||
| NC2||15||8||23||23.67 | |||
|- | |||
| NC3||15||8||23||50.85 | |||
|- | |||
| 6P2||15||8||23||493.73 | |||
|- | |||
| 6P3||15||8||23||154.56 | |||
|} |
Latest revision as of 09:07, 29 October 2014
Preparation of WGBS of cfDNA from plasma using KAPA Hyper Prep kit & NEXTflex Cell Free DNA-Seq kit[edit]
- REPEAT experiment of 2014-10-22
- KAPA Hyper Prep Kit Manual
- The two experiments were performed in parallel. I will list the procedures of KAPA Hyper Prep kit through adaptor ligation.
- I then will list the procedure of NEXTflex Cell Free DNA-Seq kit
- The down stream procedures, including bead purification of adaptor-ligated DNA, bisulfite conversion and amplification, were performed together and will be listed in the last part of this lab note.
KAPA Hyper Prep kit Experiment[edit]
Sample list
No. Sample IDs Conc. (ng/ul) #1 NC-2 0.325 #2 NC-3 0.538 #3 6P-2 1.10 #4 6P-3 1.57
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing[edit]
End repair & dA-tailing reaction mix[edit]
Components | Volume | half rxn | 5.2x rxn |
End repair/dA-tailing buffer | 7.00 | 3.50 | 18.20 |
End repair/dA-tailing enzyme mix | 3.00 | 1.50 | 7.80 |
Total | 10.00 | 5.00 |
Reaction set up[edit]
Sample IDs | Conc. (ng/ul) | Volume for 1ng (ul) | End repair/dA-tailing rxn mix | H2O (ul) | Total (ul) |
NC-2 | 0.33 | 3.08 | 5.00 | 21.92 | 30.00 |
NC-3 | 0.54 | 1.86 | 5.00 | 23.14 | 30.00 |
6P-2 | 1.10 | 0.91 | 5.00 | 24.09 | 30.00 |
6P-3 | 1.57 | 0.64 | 5.00 | 24.36 | 30.00 |
NTC | 0.00 | 0.00 | 5.00 | 25.00 | 30.00 |
- - Mix well
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation[edit]
Adaptor ligation reaction mix[edit]
Components | Volume | half rxn | 5.2x rxn |
Ligation buffer | 30.00 | 15.00 | 78.00 |
DNA ligase | 10.00 | 5.00 | 26.00 |
H2O | 8.00 | 4.00 | 20.80 |
Total | 48.00 | 24.00 |
Adaptor ligation reaction set up[edit]
Components | Volume (ul) |
End repair & dA-tailing rxn product | 30.00 |
1:20 diluted adaptor (750nM) | 1.00 |
Adaptor ligation rxn mix | 24.00 |
Total | 55.00 |
- I used 1:20 diluted Truseq adaptor prepared in June 2014
KAPA Hyper Prep indexing
Sample IDs Index NC-2 11 NC-3 12 6P-2 21 6P-3 22 NTC 11
- - Add 1ul of 1:20 diluted M-adaptor
- - Add 24ul of adaptor ligation reaction mix
- - Mix well
- - 20C, 15min
- - Continue to SPRI clean up immediately
- Final conc. of adaptor in 55ul reaction is ~13nM, which is very close to 14nM recommended by the kit.
NEXTflex Cell Free DNA Seq Kit[edit]
Sample list (the same as above)
No. Sample IDs Conc. (ng/ul) #1 NC-2 0.325 #2 NC-3 0.538 #3 6P-2 1.10 #4 6P-3 1.57
- Note: I also reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- NEXTflex Cell Free DNA Seq reaction set up (exactly in the instruction):
- - 32ul of cell-free DNA
- - 15ul of NEXTflex End-Repair & Adenylation Buffer mix
- - 3ul of NEXTflex End-Repair & Adenylation Enzyme mix
- - Total reaction: 50ul
End repair & Adenylation[edit]
End repair & Adenylation reaction mix[edit]
Components | Volume | half rxn | 5.2x rxn |
End repair & Adenylation buffer mix | 15.00 | 7.50 | 39.00 |
End repair & Adenylation enzyme mix | 3.00 | 1.50 | 7.80 |
Total | 18.00 | 9.00 |
Reaction set up[edit]
Sample IDs | Conc. (ng/ul) | Volume for 1ng (ul) | End repair/dA-tailing rxn mix | H2O (ul) | Total (ul) |
NC-2 | 0.33 | 3.08 | 9.00 | 12.92 | 25.00 |
NC-3 | 0.54 | 1.86 | 9.00 | 14.14 | 25.00 |
6P-2 | 1.10 | 0.91 | 9.00 | 15.09 | 25.00 |
6P-3 | 1.57 | 0.64 | 9.00 | 15.36 | 25.00 |
NTC | 0.00 | 0.00 | 9.00 | 16.00 | 25.00 |
- - Mix the reaction well
- - 22C, 20min
- - 72C, 20min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation[edit]
- For this kit, it suggest to dilute the 25uM adaptor 1:8 or 9X (2777.78nM) and use 2.5ul in total reaction 100ul. The final conc. of adaptor in ligation reaction is 69.4nM, which is ~5 time more adaptor concentration suggested by KAPA Hyper Prep kit.
- Truseq adaptor concentration is 15uM. I diluted it to 2777.78nM by mixing 2ul of 15uM adaptor with 8.8ul H2O and use 1.25ul in total 50ul of ligation reaction.
Adaptor ligation reaction set up[edit]
NEXTflex indexing
Sample IDs Index NC-2 6 NC-3 7 6P-2 8 6P-3 10 NTC 6
- The Enzyme mix is already in buffer, so I
- - Add 1.25ul of 2777.7nM TruSeq adaptor
- - Add 23.75ul of NEXTflex Ligase enzyme mix directly to dA-tailed DNA
- - Mix well
- - 22C, 15min
- - Continue to SPRI clean up immediately
1X AMPure bead purification[edit]
- The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
- KAPA Hyper prep kit: does 1 round of 1X AMPure bead purification
- NEXTflext: does 2 round of 1X AMPure bead purification. I guess due to the high amount of adaptor in ligation reaction (5X more than KAPA Hyper)
- Aliquot AMPure bead in 1.5mL tube
- - Add 55ul (for KAPA Hyper) or 50ul (for NEXTflex) AMPure bead. Mix by pipetting 10x
- - Sit for 15min (NEXTflex suggested 5min)
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - Dry the bead for 3-5min
- - Resuspend with 21ul EB buffer
- - For NEXTflex, I resuspended with 50ul EB and repeat 1X vol AMPure bead purification following the procedures above.
- - Continue to bisulfite conversion immediately
Bisulfite conversion[edit]
- I performed bisulfite conversion using the same procedure following manufacturer's instruction of EZ DNA Methylation-Lightning kit and elute with 32ul elution buffer.
Prep
- - Aliquot 6.12mL of binding buffer & mix with 10.2ul of 10ng/ul tRNA
- - Aliquot 5.1mL of Washing buffer
- - Aliquot 2.1mL of Desulfonation buffer
- - Aliquot 340ul of Elution buffer
Amplification[edit]
- Since from the previous experiment, I saw very low amplification by doing 1 round of PCR at high cycle number. I would follow the old optimize protocol by doing 2 round PCR. the amplification was lower than the WGBS using Thermoscientific system I did before. I would increase 1 round PCR to 15 cycle instead of 12cycles. Do 1X AMPure bead purification and continue to 2nd round PCR.
- I use amplification primers in our lab designed for TruSeq adaptor.
1st round PCR[edit]
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 11.5x rxn mix |
Bis-cvt DNA | 30.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 | 57.50 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 | 14.38 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 | 17.25 |
50X SYBG | 50 | X | 0.4 | X | 0.40 | 4.60 |
PfuTurbo Cx | 2.5 | Unit/ul | 2.5 | unit | 1.00 | 11.50 |
H2O | 10.85 | 124.78 | ||||
Total | 50.00 |
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 15 --> 72C for 2min
- - Took 3ul for PAGE verification with no bead purify
File:ZhangLab 2 2014-10-24 22hr 44min 1st-PCR-KAPA-NEXTflex-3ulPCR.jpg
- - Purify with 1X AMPure beads
- - Elute with 40ul EB Buffer
- - Continue to 2nd round PCR. No PAGE verification.
2nd round PCR (QUICK TEST to monitor PCR cycle number)[edit]
- I did a quick test in small volume by using 2ul of bead purified 1st round PCR in total 10ul. Then split into 2X of 4.5ul and stopped based on qPCR curve.
Components | Conc | unit | Final conc. | unit | Volume (ul) | 12 rxn mix |
Purified 1st round DNA | 2.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 2.00 | 24.00 |
dNTP mix | 10 | mM | 0.25 | mM | 0.25 | 3.00 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.30 | 3.60 |
50X SYBR | 50 | X | 0.4 | X | 0.08 | 0.96 |
Phusion HF | 2 | unit/ul | 0.10 | 1.20 | ||
H2O | 5.27 | 63.24 | ||||
Total | 10.00 |
- - Aliquot 8 + 2ul DNA template
- - Mix well.
- - Split 4.5ul
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5, 6, and 7 cycles --> 72C for 2min
KAPA Hyper prep
- - Sample NC2 & NC3 stopped at 5X and 7X
- - Sample 6P2 & 6P3 stopped at 5X and 6X
NEXTflex
- - Sample NC2 & NC3 stopped at 5X and 7X
- - Sample 6P2 & 6P3 stopped at 5X and 7X
- - Verify all PCR product in 6% TBE gel
File:ZhangLab 2 2014-10-27 12hr 50min 2ndPCR-KAPA-NEXTflex-5x.jpg File:ZhangLab 2 2014-10-27 12hr 50min 2ndPCR-KAPA-NEXTflex-6x-7x.jpg
- From the gel image, XXXXX
- - I do 2nd round PCR in the large volume by using all 1st round PCR as template. I reduce template amount of 6P samples by KAPA Hyper Prep kit to 10% and increase 2nd round PCR to 6 cycles. For the rest samples, I use 20% of DNA template from 1st round PCR and ran 2nd round PCR for 8 cycles.
2nd round PCR (use all 1st round PCR)[edit]
Mix 1: Low signal[edit]
- Sample: KAPA Hyper Prep, NC2 & NC3 all sample of NEXTflex
Components | Conc | unit | Final conc. | unit | Volume (ul) | 6.2 rxn mix |
Purified 1st round DNA | 36.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 36.00 | 223.20 |
dNTP mix | 10 | mM | 0.25 | mM | 4.50 | 27.90 |
TruS_F/R | 10 | uM | 0.3 | uM | 5.40 | 33.48 |
50X SYBR | 50 | X | 0.4 | X | 1.44 | 8.93 |
Phusion HF | 2 | unit/ul | 1.80 | 11.16 | ||
H2O | 94.86 | 588.13 | ||||
Total | 180.00 |
- - Aliquot 144ul + 36ul of template
- - Split 50ul to each well of 8-tube strip
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8cycles --> 72C for 2min
Mix 2: High signal[edit]
- Sample: KAPA Hyper Prep 6P2 & 6P3
Components | Conc | unit | Final conc. | unit | Volume (ul) | 2.2 rxn mix |
Purified 1st round DNA | 36.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 72.00 | 158.40 |
dNTP mix | 10 | mM | 0.25 | mM | 9.00 | 19.80 |
TruS_F/R | 10 | uM | 0.3 | uM | 10.80 | 23.76 |
50X SYBR | 50 | X | 0.4 | X | 2.88 | 6.34 |
Phusion HF | 2 | unit/ul | 3.60 | 7.92 | ||
H2O | 225.72 | 496.58 | ||||
Total | 360.00 |
- - Aliquot 324ul + 36ul of template
- - Split 50ul to each well of 8-tube strip
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 6 cycles --> 72C for 2min
- - Do 1 round (before I do 2 rounds, but this experiment the adaptor dimer was much fainter than before) bead purification of all PCR product with 1X AMPure beads and elute with 50ul EB buffer.
- - Verified 2ul of bead purified sequencing library in 6% TBE gel
File:ZhangLab 2 2014-10-27 17hr 01min 2ul PAGEverify KAPAHyper NEXTflex WGBS.jpg
- - Took 1.5ul for Qubit dsDNA HS quantification
Qubit dsDNA HS quantification result[edit]
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Conc. (ng/ul) | Yield in 46ul |
NC-2_KAPA | 6.92 | ng/mL | 1.5 | 133.33 | 0.92 | 42.44 |
NC-2_KAPA | 23.2 | ng/mL | 1.5 | 133.33 | 3.09 | 142.29 |
6P2_KAPA | 137 | ng/mL | 1.5 | 133.33 | 18.27 | 840.27 |
6P3_KAPA | 72.6 | ng/mL | 1.5 | 133.33 | 9.68 | 445.28 |
NC2_NEXTflex | 3.86 | ng/mL | 1.5 | 133.33 | 0.51 | 23.67 |
NC3_NEXTflex | 8.29 | ng/mL | 1.5 | 133.33 | 1.11 | 50.85 |
6P2_NEXTflex | 80.5 | ng/mL | 1.5 | 133.33 | 10.73 | 493.73 |
6P3_NEXTflex | 25.2 | ng/mL | 1.5 | 133.33 | 3.36 | 154.56 |
Comparison of the PCR cycle number and yield of these samples to the previous protocol using only Klenow fragment, exo-[edit]
Sample | 1st PCR | 2nd PCR | Total PCR cycles | Yield (ng) |
Klenow exo- | ||||
NC2 | 12 | 7 | 19 | 111.20 |
NC3 | 12 | 7 | 19 | 144.00 |
6P2 | 12 | 6 | 18 | 756.00 |
6P3 | 12 | 6 | 18 | 369.60 |
KAPA Hyper Prep kit | ||||
NC2 | 15 | 8 | 23 | 42.44 |
NC3 | 15 | 8 | 23 | 142.29 |
6P2 | 15 | 6 | 21 | 840.27 |
6P3 | 15 | 6 | 21 | 445.28 |
NEXTflex Cell Free DNA-Seq kit | ||||
NC2 | 15 | 8 | 23 | 23.67 |
NC3 | 15 | 8 | 23 | 50.85 |
6P2 | 15 | 8 | 23 | 493.73 |
6P3 | 15 | 8 | 23 | 154.56 |