Rui:LabNotes/Haplotyping/2014-12-2

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Purpose

  • repeat selector procedure:
  • selector : gDNA = 200:1; linker : selector = 3:1
  • direct gDNA input: 12, 24, 48ng
  • compare selector vs padlock probe
  • premade

Samples

  • HapMap gDNA from Coriell GM18506 (diluted to 50ng/ul)
  gDNA input Selector set Probe ratio Indx Test
#1 48ng linkerV2_UMI 200:1; 3:1 Indx01 selector_UMI
#2 24ng linkerV2_UMI 200:1; 3:1 Indx02 selector_UMI
#3 12ng linkerV2_UMI 200:1; 3:1 Indx03 selector_UMI
#4 NTC linkerV2_UMI 200:1; 3:1 Indx04 selector_UMI
#5 48ng linkerV2 200:1; 3:1 Indx05 selector
#6 24ng linkerV2 200:1; 3:1 Indx06 selector
#7 12ng linkerV2 200:1; 3:1 Indx07 selector
#8 NTC linkerV2 200:1; 3:1 Indx08 selector
#9 48ng linkerV2 200:1 Indx09 padlock probe
#10 24ng linkerV2 200:1 Indx10 padlock probe
#11 12ng linkerV2 200:1 Indx11 padlock probe
#12 NTC linkerV2 200:1 Indx12 padlock probe
#13 50ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx13 selector_UMI
#14 20ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx14 selector_UMI

Procedure

1a. R.E digestion

Components 48ng 24ng 12ng NTC
gDNA (12ng/ul) 4.00 2.00 1.00 0.00
10x cutsmart buffer 1.00 1.00 1.00 1.00
DpnII (50U/ul) -> dilute to 5U/ul 2.50 1.25 0.63 0.63
BfaI (10U/ul) -> dilute to 2U/ul 2.50 1.25 0.63 0.63
H2O 0.00 4.50 6.75 7.75
Total 10.00 10.00 10.00 10.00
Temperature Duration
Digestion 37C, 1hr
hold 4C
Components Volume (ul) Final conc.
10X NEBuffer 3.1 1 1x
Temperature Duration
Digestion 37C, 30min
heat inactivation 80C, 20min
hold 4C

1b. Assembly of selecotr and linker

Components	                        Vol. (ul)	Final	
LMS_selector V2 (1ng/ul: 33nM)	        20.00	        0.4ng/ul	ratio 200:1
linkerV2 or linkerV2_UMI  (100nM) 	20.00	        3:1	        linker:selector
10X AmpLigase buffer	                5.00	        1x	
H2O	                                5.00		
Total	                                50.00		
  • 94C, 2min -> 37C, 10min

2. Hybridization: Ampligase buffer needs to be aliquot and stored individually

Components Final conc. 48ng 24ng 12ng NTC 48ng 24ng 12ng NTC
digested gDNA 0/12/24/48ng 10.00 10.00 10.00 10.00 4.00 2.00 1.00 0.00
10x Ampligase buffer 1x 1.36 1.68 1.84 1.84 2.1 2.1 2.1 2.1
MgCl2 (25mM) 2mM 1.60 1.60 1.60 1.60 0.00 0.00 0.00 0.00
NAD (50mM) 1mM 0.40 0.40 0.40 0.40 0.00 0.00 0.00 0.00
Selector/linker 200:1 6.38 3.20 1.60 1.60 2.55 1.28 0.64 0.64
H2O   0.26 3.12 4.56 4.56 11.35 14.62 16.26 17.26
Total   20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
Thermocycler steps Duration
Denature 95C, 5min
Cool down to 60C slowely at -0.2C/sec
Hold 60C, 16hrs

3. Synthesis: Ampligase plus Taq pol.

Components Selector Probe
Ampligase (2.5U/ul diluted in water) 1 0.4
HemoKlen Taq (10U/ul) 0 0.4
dNTP mix (1mM) 0 0.2
Total 1 1
Thermocycler steps Duration
Synthesis 60C, 2hrs
Denature 94C, 2min
hold 8C

4. ExoM: made mixture of ExoI/III before adding into samples

Components Volume (ul) Final conc.
ExoI 1  
ExoIII 1  
Total 2  
Thermocycler steps Duration
Digestion 37C, 1hr
Denature 80C, 20min
hold 8C

5. PCR amplification: quick run with 1/10 of sample for an estimation of capture efficiency

  • Sample PCR with Indexes
Components Volumn (ul) Final conc.
cirDNA template 22 all
Q5 MM 30 1x
10 uM CP-2-RA 1.2 0.2uM
10 uM AmpR6.3.Indx 1.2 0.2uM
20x SYBR 0.5 0.2x
H2O 5.09999999999999  
Total 60  
Realtime-PCR profile QPCR cycles
98C 5min 1
98C 15sec -> 58C 30sec -> 72C 15sec 8
98C 15sec -> 72C 15sec 16
72C 3min 1
Hold 10C  

6. Beads purfication on PCR amplicons

Components Volume (ul) Final conc.
PCR amplicon 55 5ul for gel
AMPure beads 50 0.8X
* Clearly remove majority of bands/smear below 200bp    

Results

  • QPCR curve

File:Nv4 1-4 QPCR.jpgFile:Nv4 5-12 QPCR.jpg

  • PAGE check

File:Nv4 1-4 gel.jpgFile:Nv4 5-12 gel.jpg

Sequence libraries

  • Following libraries were combined and size selected for sequencing
  • Tube label: RL-Nv4_UMI-Dec3
  gDNA input Selector set Probe ratio Indx Test Mixed V (ul)
#1 48ng linkerV2_UMI 200:1; 3:1 Indx01 selector_UMI 2
#2 24ng linkerV2_UMI 200:1; 3:1 Indx02 selector_UMI 4
#3 12ng linkerV2_UMI 200:1; 3:1 Indx03 selector_UMI 8
#13 50ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx13 selector_UMI 2
#14 20ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx14 selector_UMI 4
* size selection can be skipped, pretty clean below 200bp            
  • Tube label: RL-Nv4-Dec3
  gDNA input Selector set Probe ratio Indx Test Mixed V (ul)
#5 48ng linkerV2 200:1; 3:1 Indx05 selector 2
#6 24ng linkerV2 200:1; 3:1 Indx06 selector 4
#7 12ng linkerV2 200:1; 3:1 Indx07 selector 8
#9 48ng linkerV2 200:1 Indx09 padlock probe 2
#10 24ng linkerV2 200:1 Indx10 padlock probe 4
#11 12ng linkerV2 200:1 Indx11 padlock probe 8
* need size selection, still have a few bands below 200bp            

Correction of barcode assignment.

Sample_ID Seq. read ID Sample_Name I7_Index_ID index CorrectedID index
RL-Nv3-Dec3#2 RL-Nv3-Dec3-2 12ng_1a-SNPs A018 AGTTCA A002 CGATGT
RL-Nv3-Dec3#3 RL-Nv3-Dec3-3 12ng_1b-SNPs A019 GACGCA A003 TTAGGC
RL-Nv3-Dec3#4 RL-Nv3-Dec3-4 12ng_1c-SNPs A020 CCTGTC A004 TGACCA
RL-Nv3-Dec3#5 RL-Nv3-Dec3-5 12ng_3a-SNPs A021 CAACCC A005 ACAGTG
RL-Nv3-Dec3#6 RL-Nv3-Dec3-6 12ng_3b-SNPs A022 CTCGGA A006 GCCAAT
RL-Nv3-Dec3#7 RL-Nv3-Dec3-7 12ng_3c-SNPs A023 TCGAAA A007 CAGATC
RL-Nv3-Dec3#8 RL-Nv3-Dec3-8 12ng_5a-SNPs A024 ATTCGC A008 ACTTGA
RL-Nv3-Dec3#9 RL-Nv3-Dec3-9 12ng_5b-SNPs A025 TACTGC A009 GATCAG
RL-Nv3-Dec3#10 RL-Nv3-Dec3-10 12ng_5c-SNPs A026 TCGTGA A010 TAGCTT
RL-Nv3-Dec3#11 RL-Nv3-Dec3-11 12ng_7a-SNPs A027 TACGCG A011 GGCTAC
RL-Nv3-Dec3#12 RL-Nv3-Dec3-12 12ng_7b-SNPs A028 AGGTGC A012 CTTGTA
RL-Nv3-Dec3#13 RL-Nv3-Dec3-13 12ng_7c-SNPs A029 ACGAAC A013 CCATGA
RL-Nv3-Dec3#14 RL-Nv3-Dec3-14 12ng_10a-SNPs A030 TTAGTG A014 AAGACA
RL-Nv3-Dec3#15 RL-Nv3-Dec3-15 12ng_10b-SNPs A031 CACCAC A015 CTTCCT
RL-Nv3-Dec3#16 RL-Nv3-Dec3-16 12ng_10c-SNPs A032 GCAAGG A016 GGGGTT
RL-Nv4_UMI-Dec3#1 RL-Nv4_UMI-Dec3-1 48ng-selector_UMI A001 ATCACG A017 CTCATC
RL-Nv4_UMI-Dec3#2 RL-Nv4_UMI-Dec3-2 24ng-selector_UMI A002 CGATGT A018 AGTTCA
RL-Nv4_UMI-Dec3#3 RL-Nv4_UMI-Dec3-3 12ng-selector_UMI A003 TTAGGC A019 GACGCA
RL-Nv4_UMI-Dec3#13 RL-Nv4_UMI-Dec3-13 50ng_digst-selector_UMI A013 CCATGA A029 ACGAAC
RL-Nv4_UMI-Dec3#14 RL-Nv4_UMI-Dec3-14 20ng_digst-selector_UMI A014 AAGACA A030 TTAGTG
RL-Nv4-Dec3#5 RL-Nv4-Dec3-5 48ng-selector A005 ACAGTG A021 CAACCC
RL-Nv4-Dec3#6 RL-Nv4-Dec3-6 24ng-selector A006 GCCAAT A022 CTCGGA
RL-Nv4-Dec3#7 RL-Nv4-Dec3-7 12ng-selector A007 CAGATC A023 TCGAAA
RL-Nv4-Dec3#9 RL-Nv4-Dec3-9 48ng-selector A009 GATCAG A025 TACTGC
RL-Nv4-Dec3#10 RL-Nv4-Dec3-10 24ng-selector A010 TAGCTT A026 TCGTGA
RL-Nv4-Dec3#11 RL-Nv4-Dec3-11 12ng-selector A011 GGCTAC A027 TACGCG