Rui:LabNotes/Haplotyping/2014-12-2: Difference between revisions
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===1b. Assembly of selecotr and linker === | ===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=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:10pt" | |||
|style="background-color:#D9D9D9;font-weight:bold" width="192" height="15" | Components | |||
|style="background-color:#D9D9D9;font-weight:bold" width="72" align="center" | Final conc. | |||
|style="background-color:#CCFFCC" width="72" valign="bottom" | 48ng | |||
|style="background-color:#CCFFCC" width="79" valign="bottom" | 24ng | |||
|style="background-color:#CCFFCC" width="79" valign="bottom" | 12ng | |||
|style="background-color:#CCFFCC" width="63" valign="bottom" | NTC | |||
|style="background-color:#CCFFCC" width="78" valign="bottom" | 48ng | |||
|style="background-color:#CCFFCC" width="78" valign="bottom" | 24ng | |||
|style="background-color:#CCFFCC" width="78" valign="bottom" | 12ng | |||
|style="background-color:#CCFFCC" width="78" valign="bottom" | NTC | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | digested gDNA | |||
| align="center" | 0/12/24/48ng | |||
| align="center" align="right" valign="bottom" | 10.00 | |||
| align="center" align="right" valign="bottom" | 10.00 | |||
| align="center" align="right" valign="bottom" | 10.00 | |||
| align="center" align="right" valign="bottom" | 10.00 | |||
| align="center" align="right" valign="bottom" | 4.00 | |||
| align="center" align="right" valign="bottom" | 2.00 | |||
| align="center" align="right" valign="bottom" | 1.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
|- style="font-size:10pt" | |||
| height="15" | 10x Ampligase buffer | |||
| align="center" | 1x | |||
| align="center" align="right" valign="bottom" | 1.36 | |||
| align="center" align="right" valign="bottom" | 1.68 | |||
| align="center" align="right" valign="bottom" | 1.84 | |||
| align="center" align="right" valign="bottom" | 1.84 | |||
| align="center" valign="bottom" | 2.1 | |||
| align="center" valign="bottom" | 2.1 | |||
| align="center" valign="bottom" | 2.1 | |||
| align="center" valign="bottom" | 2.1 | |||
|- style="font-size:10pt" | |||
| height="15" | MgCl2 (25mM) | |||
| align="center" | 2mM | |||
| align="center" align="right" valign="bottom" | 1.60 | |||
| align="center" align="right" valign="bottom" | 1.60 | |||
| align="center" align="right" valign="bottom" | 1.60 | |||
| align="center" align="right" valign="bottom" | 1.60 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | NAD (50mM) | |||
| align="center" valign="bottom" | 1mM | |||
| align="center" align="right" valign="bottom" | 0.40 | |||
| align="center" align="right" valign="bottom" | 0.40 | |||
| align="center" align="right" valign="bottom" | 0.40 | |||
| align="center" align="right" valign="bottom" | 0.40 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
| align="center" align="right" valign="bottom" | 0.00 | |||
|- style="font-size:10pt" | |||
|style="color:#FF0000" height="15" valign="bottom" | Selector/linker | |||
| align="center" valign="bottom" | 200:1 | |||
| align="center" align="right" | 6.38 | |||
| align="center" align="right" | 3.20 | |||
| align="center" align="right" | 1.60 | |||
| align="center" align="right" | 1.60 | |||
| align="center" valign="bottom" | 2.55 | |||
| align="center" valign="bottom" | 1.28 | |||
| align="center" valign="bottom" | 0.64 | |||
| align="center" valign="bottom" | 0.64 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | H2O | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" valign="bottom" | 0.26 | |||
| align="center" valign="bottom" | 3.12 | |||
| align="center" valign="bottom" | 4.56 | |||
| align="center" valign="bottom" | 4.56 | |||
| align="center" valign="bottom" | 11.35 | |||
| align="center" valign="bottom" | 14.62 | |||
| align="center" valign="bottom" | 16.26 | |||
| align="center" valign="bottom" | 17.26 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | Total | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
| align="center" align="right" valign="bottom" | 20.00 | |||
|} | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Thermocycler steps | |||
| width="72" align="center" valign="bottom" | Duration | |||
|- style="font-size:10pt" | |||
| height="15" | Denature | |||
| 95C, 5min | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | Cool down to 60C slowely | |||
| at -0.2C/sec | |||
|- style="font-size:10pt" | |||
| height="15" | Hold | |||
| 60C, 16hrs | |||
|} | |||
===3. Synthesis: Ampligase plus Taq pol.=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#CCFFCC;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Components | |||
| width="72" align="center" | Selector | |||
| width="72" align="center" | Probe | |||
|- style="font-size:10pt" | |||
|style="color:#FF0000" height="15" valign="bottom" | Ampligase (2.5U/ul diluted in water) | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 0.4 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | HemoKlen Taq (10U/ul) | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0.4 | |||
|- style="font-size:10pt" | |||
| height="15" | dNTP mix (1mM) | |||
| align="center" align="center" | 0 | |||
| align="center" align="center" valign="top" | 0.2 | |||
|- style="font-size:10pt" | |||
| height="15" | Total | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" valign="top" | 1 | |||
|} | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Thermocycler steps | |||
| width="72" align="center" valign="bottom" | Duration | |||
|- style="font-size:10pt" | |||
| height="15" | Synthesis | |||
| 60C, 2hrs | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | Denature | |||
| 94C, 2min | |||
|- style="font-size:10pt" | |||
| height="15" | hold | |||
| 8C | |||
|} | |||
===4. ExoM: made mixture of ExoI/III before adding into samples=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Components | |||
| width="72" align="center" | Volume (ul) | |||
| width="72" align="center" | Final conc. | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | ExoI | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | | |||
|- style="font-size:10pt" | |||
| height="15" | ExoIII | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | | |||
|- style="font-size:10pt" | |||
| height="15" | Total | |||
| align="center" align="center" | 2 | |||
| align="center" valign="top" | | |||
|} | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Thermocycler steps | |||
| width="72" align="center" valign="bottom" | Duration | |||
|- style="font-size:10pt" | |||
| height="15" | Digestion | |||
| 37C, 1hr | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | Denature | |||
| 80C, 20min | |||
|- style="font-size:10pt" | |||
| height="15" | hold | |||
| 8C | |||
|} | |||
===5. PCR amplification: quick run with 1/10 of sample for an estimation of capture efficiency=== | |||
* Sample PCR with Indexes | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt" | |||
| width="192" height="15" | Components | |||
| width="72" | Volumn (ul) | |||
|style="font-weight:bold" width="72" align="center" | Final conc. | |||
|- style="font-size:10pt" | |||
| height="15" | cirDNA template | |||
| align="center" | 22 | |||
| all | |||
|- style="font-size:10pt" | |||
| height="15" | Q5 MM | |||
| align="center" | 30 | |||
| 1x | |||
|- style="font-size:10pt" | |||
| height="15" | 10 uM CP-2-RA | |||
| align="center" | 1.2 | |||
| 0.2uM | |||
|- style="font-size:10pt" | |||
| height="15" | 10 uM AmpR6.3.Indx | |||
| align="center" | 1.2 | |||
| 0.2uM | |||
|- style="font-size:10pt" | |||
| height="15" | 20x SYBR | |||
| align="center" | 0.5 | |||
| 0.2x | |||
|- style="font-size:10pt" | |||
| height="15" | H2O | |||
| align="center" | 5.09999999999999 | |||
| align="center" | | |||
|- style="font-size:10pt" | |||
| height="15" | Total | |||
| align="center" | 60 | |||
| align="center" | | |||
|} | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Realtime-PCR profile | |||
| width="72" align="center" valign="bottom" | QPCR cycles | |||
|- style="font-size:10pt" | |||
| height="15" | 98C 5min | |||
| align="center" | 1 | |||
|- style="font-size:10pt" | |||
| height="15" | 98C 15sec -> 58C 30sec -> 72C 15sec | |||
| align="center" | 8 | |||
|- style="font-size:10pt" | |||
| height="15" | 98C 15sec -> 72C 15sec | |||
| align="center" | 16 | |||
|- style="font-size:10pt" | |||
| height="15" | 72C 3min | |||
| align="center" | 1 | |||
|- style="font-size:10pt" | |||
| height="15" | Hold 10C | |||
| align="center" | | |||
|} | |||
===6. Beads purfication on PCR amplicons=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt;font-weight:bold" | |||
| width="192" height="15" | Components | |||
| width="72" align="center" | Volume (ul) | |||
| width="72" align="center" | Final conc. | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | PCR amplicon | |||
| align="center" align="center" | 55 | |||
| align="center" | 5ul for gel | |||
|- style="font-size:10pt" | |||
| height="15" | AMPure beads | |||
| align="center" align="center" | 50 | |||
| align="center" | 0.8X | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | * Clearly remove majority of bands/smear below 200bp | |||
| align="center" | | |||
| align="center" | | |||
|} | |||
==Results== | |||
* QPCR curve | |||
[[File:Nv4_1-4_QPCR.jpg|400px]][[File:Nv4_5-12_QPCR.jpg|400px]] | |||
* PAGE check | |||
[[File:Nv4_1-4_gel.jpg|400px]][[File:Nv4_5-12_gel.jpg|400px]] | |||
==Sequence libraries== | |||
* Following libraries were combined and size selected for sequencing | |||
* Tube label: '''RL-Nv4_UMI-Dec3''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#CCFFCC;font-size:10pt" valign="bottom" | |||
| align="center" width="77" height="15" | | |||
| width="192" | gDNA input | |||
| width="72" | Selector set | |||
| width="72" | Probe ratio | |||
| width="79" | Indx | |||
| width="79" | Test | |||
| width="63" | Mixed V (ul) | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #1 | |||
| valign="bottom" | 48ng | |||
| valign="bottom" | linkerV2_UMI | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx01 | |||
| valign="bottom" | selector_UMI | |||
| align="center" valign="bottom" | 2 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #2 | |||
| valign="bottom" | 24ng | |||
| valign="bottom" | linkerV2_UMI | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx02 | |||
| valign="bottom" | selector_UMI | |||
| align="center" valign="bottom" | 4 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #3 | |||
| valign="bottom" | 12ng | |||
| valign="bottom" | linkerV2_UMI | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx03 | |||
| valign="bottom" | selector_UMI | |||
| align="center" valign="bottom" | 8 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #13 | |||
| valign="bottom" | 50ng_digested (Kun's, asPC) | |||
| valign="bottom" | linkerV2_UMI | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx13 | |||
| valign="bottom" | selector_UMI | |||
| align="center" valign="bottom" | 2 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #14 | |||
| valign="bottom" | 20ng_digested (Kun's, asPC) | |||
| valign="bottom" | linkerV2_UMI | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx14 | |||
| valign="bottom" | selector_UMI | |||
| align="center" valign="bottom" | 4 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | * size selection can be skipped, pretty clean below 200bp | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|} | |||
* Tube label: '''RL-Nv4-Dec3''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#CCFFCC;font-size:10pt" valign="bottom" | |||
| align="center" width="77" height="15" | | |||
| width="192" | gDNA input | |||
| width="72" | Selector set | |||
| width="72" | Probe ratio | |||
| width="79" | Indx | |||
| width="79" | Test | |||
| width="63" | Mixed V (ul) | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #5 | |||
| valign="bottom" | 48ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx05 | |||
| valign="bottom" | selector | |||
| align="center" valign="bottom" | 2 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #6 | |||
| valign="bottom" | 24ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx06 | |||
| valign="bottom" | selector | |||
| align="center" valign="bottom" | 4 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #7 | |||
| valign="bottom" | 12ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1; 3:1 | |||
| valign="bottom" | Indx07 | |||
| valign="bottom" | selector | |||
| align="center" valign="bottom" | 8 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #9 | |||
| valign="bottom" | 48ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1 | |||
| valign="bottom" | Indx09 | |||
| valign="bottom" | padlock probe | |||
| align="center" valign="bottom" | 2 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #10 | |||
| valign="bottom" | 24ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1 | |||
| valign="bottom" | Indx10 | |||
| valign="bottom" | padlock probe | |||
| align="center" valign="bottom" | 4 | |||
|- style="font-size:10pt" | |||
| height="15" valign="bottom" | #11 | |||
| valign="bottom" | 12ng | |||
| valign="bottom" | linkerV2 | |||
| align="center" valign="bottom" | 200:1 | |||
| valign="bottom" | Indx11 | |||
| valign="bottom" | padlock probe | |||
| align="center" valign="bottom" | 8 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="15" | * need size selection, still have a few bands below 200bp | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|} | |||
===Correction of barcode assignment.=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:10pt" valign="bottom" | |||
| width="129" height="15" | Seq. read ID | |||
| width="139" | Sample | |||
|style="background-color:#D9D9D9" width="103" | wrong I7_Index_ID | |||
|style="background-color:#D9D9D9" width="79" | index | |||
|style="background-color:#FFFF00" width="65" | CorrectedID | |||
|style="background-color:#FFFF00" width="65" | index | |||
| width="129" | Old read ID | |||
|style="background-color:#FFFF00" width="124" | Corrected read ID | |||
| width="65" | real index | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-2 | |||
| 12ng_1a-SNPs | |||
|style="background-color:#D9D9D9" | A018 | |||
|style="background-color:#D9D9D9" | AGTTCA | |||
|style="background-color:#FFFF00" | A002 | |||
|style="background-color:#FFFF00" | CGATGT | |||
| RL-Nv3-Dec3-2 | |||
|style="background-color:#FFFF00" | RL-Nv4_UMI-Dec3-2 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-3 | |||
| 12ng_1b-SNPs | |||
|style="background-color:#D9D9D9" | A019 | |||
|style="background-color:#D9D9D9" | GACGCA | |||
|style="background-color:#FFFF00" | A003 | |||
|style="background-color:#FFFF00" | TTAGGC | |||
| RL-Nv3-Dec3-3 | |||
|style="background-color:#FFFF00" | RL-Nv4_UMI-Dec3-3 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-4 | |||
| 12ng_1c-SNPs | |||
|style="background-color:#D9D9D9" | A020 | |||
|style="background-color:#D9D9D9" | CCTGTC | |||
|style="background-color:#FFFF00" | A004 | |||
|style="background-color:#FFFF00" | TGACCA | |||
| RL-Nv3-Dec3-4 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A004 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-5 | |||
| 12ng_3a-SNPs | |||
|style="background-color:#D9D9D9" | A021 | |||
|style="background-color:#D9D9D9" | CAACCC | |||
|style="background-color:#FFFF00" | A005 | |||
|style="background-color:#FFFF00" | ACAGTG | |||
| RL-Nv3-Dec3-5 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-5 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-6 | |||
| 12ng_3b-SNPs | |||
|style="background-color:#D9D9D9" | A022 | |||
|style="background-color:#D9D9D9" | CTCGGA | |||
|style="background-color:#FFFF00" | A006 | |||
|style="background-color:#FFFF00" | GCCAAT | |||
| RL-Nv3-Dec3-6 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-6 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-7 | |||
| 12ng_3c-SNPs | |||
|style="background-color:#D9D9D9" | A023 | |||
|style="background-color:#D9D9D9" | TCGAAA | |||
|style="background-color:#FFFF00" | A007 | |||
|style="background-color:#FFFF00" | CAGATC | |||
| RL-Nv3-Dec3-7 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-7 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-8 | |||
| 12ng_5a-SNPs | |||
|style="background-color:#D9D9D9" | A024 | |||
|style="background-color:#D9D9D9" | ATTCGC | |||
|style="background-color:#FFFF00" | A008 | |||
|style="background-color:#FFFF00" | ACTTGA | |||
| RL-Nv3-Dec3-8 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A008 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-9 | |||
| 12ng_5b-SNPs | |||
|style="background-color:#D9D9D9" | A025 | |||
|style="background-color:#D9D9D9" | TACTGC | |||
|style="background-color:#FFFF00" | A009 | |||
|style="background-color:#FFFF00" | GATCAG | |||
| RL-Nv3-Dec3-9 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-9 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-10 | |||
| 12ng_5c-SNPs | |||
|style="background-color:#D9D9D9" | A026 | |||
|style="background-color:#D9D9D9" | TCGTGA | |||
|style="background-color:#FFFF00" | A010 | |||
|style="background-color:#FFFF00" | TAGCTT | |||
| RL-Nv3-Dec3-10 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-10 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-11 | |||
| 12ng_7a-SNPs | |||
|style="background-color:#D9D9D9" | A027 | |||
|style="background-color:#D9D9D9" | TACGCG | |||
|style="background-color:#FFFF00" | A011 | |||
|style="background-color:#FFFF00" | GGCTAC | |||
| RL-Nv3-Dec3-11 | |||
|style="background-color:#FFFF00" | RL-Nv4-Dec3-11 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-12 | |||
| 12ng_7b-SNPs | |||
|style="background-color:#D9D9D9" | A028 | |||
|style="background-color:#D9D9D9" | AGGTGC | |||
|style="background-color:#FFFF00" | A012 | |||
|style="background-color:#FFFF00" | CTTGTA | |||
| RL-Nv3-Dec3-12 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A012 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-13 | |||
| 12ng_7c-SNPs | |||
|style="background-color:#D9D9D9" | A029 | |||
|style="background-color:#D9D9D9" | ACGAAC | |||
|style="background-color:#FFFF00" | A013 | |||
|style="background-color:#FFFF00" | CCATGA | |||
| RL-Nv3-Dec3-13 | |||
|style="background-color:#FFFF00" | RL-Nv4_UMI-Dec3-13 | |||
|style="color:#FF0000" align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-14 | |||
| 12ng_10a-SNPs | |||
|style="background-color:#D9D9D9" | A030 | |||
|style="background-color:#D9D9D9" | TTAGTG | |||
|style="background-color:#FFFF00" | A014 | |||
|style="background-color:#FFFF00" | AAGACA | |||
| RL-Nv3-Dec3-14 | |||
|style="background-color:#FFFF00" | RL-Nv4_UMI-Dec3-14 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-15 | |||
| 12ng_10b-SNPs | |||
|style="background-color:#D9D9D9" | A031 | |||
|style="background-color:#D9D9D9" | CACCAC | |||
|style="background-color:#FFFF00" | A015 | |||
|style="background-color:#FFFF00" | CTTCCT | |||
| RL-Nv3-Dec3-15 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A015 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv3-Dec3-16 | |||
| 12ng_10c-SNPs | |||
|style="background-color:#D9D9D9" | A032 | |||
|style="background-color:#D9D9D9" | GCAAGG | |||
|style="background-color:#FFFF00" | A016 | |||
|style="background-color:#FFFF00" | GGGGTT | |||
| RL-Nv3-Dec3-16 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A016 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4_UMI-Dec3-1 | |||
| 48ng-selector_UMI | |||
|style="background-color:#D9D9D9" | A001 | |||
|style="background-color:#D9D9D9" | ATCACG | |||
|style="background-color:#FFFF00" | A017 | |||
|style="background-color:#FFFF00" | CTCATC | |||
| RL-Nv4_UMI-Dec3-1 | |||
|style="background-color:#FFFF00;color:#FF0000" align="center" | | |||
|style="color:#FF0000" | A017 | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4_UMI-Dec3-2 | |||
| 24ng-selector_UMI | |||
|style="background-color:#D9D9D9" | A002 | |||
|style="background-color:#D9D9D9" | CGATGT | |||
|style="background-color:#FFFF00" | A018 | |||
|style="background-color:#FFFF00" | AGTTCA | |||
| RL-Nv4_UMI-Dec3-2 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-2 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4_UMI-Dec3-3 | |||
| 12ng-selector_UMI | |||
|style="background-color:#D9D9D9" | A003 | |||
|style="background-color:#D9D9D9" | TTAGGC | |||
|style="background-color:#FFFF00" | A019 | |||
|style="background-color:#FFFF00" | GACGCA | |||
| RL-Nv4_UMI-Dec3-3 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-3 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4_UMI-Dec3-13 | |||
| 50ng_digst-selector_UMI | |||
|style="background-color:#D9D9D9" | A013 | |||
|style="background-color:#D9D9D9" | CCATGA | |||
|style="background-color:#FFFF00" | A029 | |||
|style="background-color:#FFFF00" | ACGAAC | |||
| RL-Nv4_UMI-Dec3-13 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-13 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4_UMI-Dec3-14 | |||
| 20ng_digst-selector_UMI | |||
|style="background-color:#D9D9D9" | A014 | |||
|style="background-color:#D9D9D9" | AAGACA | |||
|style="background-color:#FFFF00" | A030 | |||
|style="background-color:#FFFF00" | TTAGTG | |||
| RL-Nv4_UMI-Dec3-14 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-14 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-5 | |||
| 48ng-selector | |||
|style="background-color:#D9D9D9" | A005 | |||
|style="background-color:#D9D9D9" | ACAGTG | |||
|style="background-color:#FFFF00" | A021 | |||
|style="background-color:#FFFF00" | CAACCC | |||
| RL-Nv4-Dec3-5 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-5 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-6 | |||
| 24ng-selector | |||
|style="background-color:#D9D9D9" | A006 | |||
|style="background-color:#D9D9D9" | GCCAAT | |||
|style="background-color:#FFFF00" | A022 | |||
|style="background-color:#FFFF00" | CTCGGA | |||
| RL-Nv4-Dec3-6 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-6 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-7 | |||
| 12ng-selector | |||
|style="background-color:#D9D9D9" | A007 | |||
|style="background-color:#D9D9D9" | CAGATC | |||
|style="background-color:#FFFF00" | A023 | |||
|style="background-color:#FFFF00" | TCGAAA | |||
| RL-Nv4-Dec3-7 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-7 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-9 | |||
| 48ng-selector | |||
|style="background-color:#D9D9D9" | A009 | |||
|style="background-color:#D9D9D9" | GATCAG | |||
|style="background-color:#FFFF00" | A025 | |||
|style="background-color:#FFFF00" | TACTGC | |||
| RL-Nv4-Dec3-9 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-9 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-10 | |||
| 24ng-selector | |||
|style="background-color:#D9D9D9" | A010 | |||
|style="background-color:#D9D9D9" | TAGCTT | |||
|style="background-color:#FFFF00" | A026 | |||
|style="background-color:#FFFF00" | TCGTGA | |||
| RL-Nv4-Dec3-10 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-10 | |||
| align="center" | | |||
|- style="font-size:10pt" valign="bottom" | |||
| height="16" | RL-Nv4-Dec3-11 | |||
| 12ng-selector | |||
|style="background-color:#D9D9D9" | A011 | |||
|style="background-color:#D9D9D9" | GGCTAC | |||
|style="background-color:#FFFF00" | A027 | |||
|style="background-color:#FFFF00" | TACGCG | |||
| RL-Nv4-Dec3-11 | |||
|style="background-color:#FFFF00" | RL-Nv3-Dec3-11 | |||
| align="center" | | |||
|} |
Latest revision as of 07:12, 8 December 2014
Purpose[edit]
- repeat selector procedure:
- selector : gDNA = 200:1; linker : selector = 3:1
- direct gDNA input: 12, 24, 48ng
- compare selector vs padlock probe
- premade
Samples[edit]
- 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[edit]
1a. R.E digestion[edit]
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[edit]
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[edit]
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.[edit]
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[edit]
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[edit]
- 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[edit]
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[edit]
- 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[edit]
- 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.[edit]
Seq. read ID | Sample | wrong I7_Index_ID | index | CorrectedID | index | Old read ID | Corrected read ID | real index |
RL-Nv3-Dec3-2 | 12ng_1a-SNPs | A018 | AGTTCA | A002 | CGATGT | RL-Nv3-Dec3-2 | RL-Nv4_UMI-Dec3-2 | |
RL-Nv3-Dec3-3 | 12ng_1b-SNPs | A019 | GACGCA | A003 | TTAGGC | RL-Nv3-Dec3-3 | RL-Nv4_UMI-Dec3-3 | |
RL-Nv3-Dec3-4 | 12ng_1c-SNPs | A020 | CCTGTC | A004 | TGACCA | RL-Nv3-Dec3-4 | A004 | |
RL-Nv3-Dec3-5 | 12ng_3a-SNPs | A021 | CAACCC | A005 | ACAGTG | RL-Nv3-Dec3-5 | RL-Nv4-Dec3-5 | |
RL-Nv3-Dec3-6 | 12ng_3b-SNPs | A022 | CTCGGA | A006 | GCCAAT | RL-Nv3-Dec3-6 | RL-Nv4-Dec3-6 | |
RL-Nv3-Dec3-7 | 12ng_3c-SNPs | A023 | TCGAAA | A007 | CAGATC | RL-Nv3-Dec3-7 | RL-Nv4-Dec3-7 | |
RL-Nv3-Dec3-8 | 12ng_5a-SNPs | A024 | ATTCGC | A008 | ACTTGA | RL-Nv3-Dec3-8 | A008 | |
RL-Nv3-Dec3-9 | 12ng_5b-SNPs | A025 | TACTGC | A009 | GATCAG | RL-Nv3-Dec3-9 | RL-Nv4-Dec3-9 | |
RL-Nv3-Dec3-10 | 12ng_5c-SNPs | A026 | TCGTGA | A010 | TAGCTT | RL-Nv3-Dec3-10 | RL-Nv4-Dec3-10 | |
RL-Nv3-Dec3-11 | 12ng_7a-SNPs | A027 | TACGCG | A011 | GGCTAC | RL-Nv3-Dec3-11 | RL-Nv4-Dec3-11 | |
RL-Nv3-Dec3-12 | 12ng_7b-SNPs | A028 | AGGTGC | A012 | CTTGTA | RL-Nv3-Dec3-12 | A012 | |
RL-Nv3-Dec3-13 | 12ng_7c-SNPs | A029 | ACGAAC | A013 | CCATGA | RL-Nv3-Dec3-13 | RL-Nv4_UMI-Dec3-13 | |
RL-Nv3-Dec3-14 | 12ng_10a-SNPs | A030 | TTAGTG | A014 | AAGACA | RL-Nv3-Dec3-14 | RL-Nv4_UMI-Dec3-14 | |
RL-Nv3-Dec3-15 | 12ng_10b-SNPs | A031 | CACCAC | A015 | CTTCCT | RL-Nv3-Dec3-15 | A015 | |
RL-Nv3-Dec3-16 | 12ng_10c-SNPs | A032 | GCAAGG | A016 | GGGGTT | RL-Nv3-Dec3-16 | A016 | |
RL-Nv4_UMI-Dec3-1 | 48ng-selector_UMI | A001 | ATCACG | A017 | CTCATC | RL-Nv4_UMI-Dec3-1 | A017 | |
RL-Nv4_UMI-Dec3-2 | 24ng-selector_UMI | A002 | CGATGT | A018 | AGTTCA | RL-Nv4_UMI-Dec3-2 | RL-Nv3-Dec3-2 | |
RL-Nv4_UMI-Dec3-3 | 12ng-selector_UMI | A003 | TTAGGC | A019 | GACGCA | RL-Nv4_UMI-Dec3-3 | RL-Nv3-Dec3-3 | |
RL-Nv4_UMI-Dec3-13 | 50ng_digst-selector_UMI | A013 | CCATGA | A029 | ACGAAC | RL-Nv4_UMI-Dec3-13 | RL-Nv3-Dec3-13 | |
RL-Nv4_UMI-Dec3-14 | 20ng_digst-selector_UMI | A014 | AAGACA | A030 | TTAGTG | RL-Nv4_UMI-Dec3-14 | RL-Nv3-Dec3-14 | |
RL-Nv4-Dec3-5 | 48ng-selector | A005 | ACAGTG | A021 | CAACCC | RL-Nv4-Dec3-5 | RL-Nv3-Dec3-5 | |
RL-Nv4-Dec3-6 | 24ng-selector | A006 | GCCAAT | A022 | CTCGGA | RL-Nv4-Dec3-6 | RL-Nv3-Dec3-6 | |
RL-Nv4-Dec3-7 | 12ng-selector | A007 | CAGATC | A023 | TCGAAA | RL-Nv4-Dec3-7 | RL-Nv3-Dec3-7 | |
RL-Nv4-Dec3-9 | 48ng-selector | A009 | GATCAG | A025 | TACTGC | RL-Nv4-Dec3-9 | RL-Nv3-Dec3-9 | |
RL-Nv4-Dec3-10 | 24ng-selector | A010 | TAGCTT | A026 | TCGTGA | RL-Nv4-Dec3-10 | RL-Nv3-Dec3-10 | |
RL-Nv4-Dec3-11 | 12ng-selector | A011 | GGCTAC | A027 | TACGCG | RL-Nv4-Dec3-11 | RL-Nv3-Dec3-11 |