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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) {| 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 |- 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 |- 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 |- 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 |- style="font-size:10pt" | height="15" valign="bottom" | #4 | valign="bottom" | NTC | valign="bottom" | linkerV2_UMI | align="center" valign="bottom" | 200:1; 3:1 | valign="bottom" | Indx04 | valign="bottom" | selector_UMI |- 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 |- 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 |- 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 |- style="font-size:10pt" | height="15" valign="bottom" | #8 | valign="bottom" | NTC | valign="bottom" | linkerV2 | align="center" valign="bottom" | 200:1; 3:1 | valign="bottom" | Indx08 | valign="bottom" | selector |- 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 |- 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 |- 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 |- style="font-size:10pt" | height="15" valign="bottom" | #12 | valign="bottom" | NTC | valign="bottom" | linkerV2 | align="center" valign="bottom" | 200:1 | valign="bottom" | Indx12 | valign="bottom" | padlock probe |- 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 |- 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 |} ==Procedure== ===1a. R.E digestion=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="background-color:#CCFFCC;font-size:10pt" |style="font-weight:bold" width="192" height="15" | Components | width="72" | 48ng | width="72" | 24ng | width="79" | 12ng | width="79" | NTC |- style="font-size:10pt" | height="15" valign="bottom" | gDNA (12ng/ul) | 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 cutsmart buffer | align="center" align="right" valign="bottom" | 1.00 | align="center" align="right" valign="bottom" | 1.00 | align="center" align="right" valign="bottom" | 1.00 | align="center" align="right" valign="bottom" | 1.00 |- style="font-size:10pt" |style="color:#FF0000" height="15" | DpnII (50U/ul) -> dilute to 5U/ul | align="center" align="right" valign="bottom" | 2.50 | align="center" align="right" valign="bottom" | 1.25 | align="center" align="right" valign="bottom" | 0.63 | align="center" align="right" valign="bottom" | 0.63 |- style="font-size:10pt" |style="color:#FF0000" height="15" valign="bottom" | BfaI (10U/ul) -> dilute to 2U/ul | align="center" align="right" valign="bottom" | 2.50 | align="center" align="right" valign="bottom" | 1.25 | align="center" align="right" valign="bottom" | 0.63 | align="center" align="right" valign="bottom" | 0.63 |- style="font-size:10pt" | height="15" valign="bottom" | H2O | align="center" align="right" | 0.00 | align="center" align="right" | 4.50 | align="center" align="right" | 6.75 | align="center" align="right" | 7.75 |- style="font-size:10pt" | height="15" valign="bottom" | Total | align="center" align="right" | 10.00 | align="center" align="right" | 10.00 | align="center" align="right" | 10.00 | align="center" align="right" | 10.00 |} {| 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" | Temperature | width="72" align="center" valign="bottom" | Duration |- style="font-size:10pt" | height="15" | Digestion | 37C, 1hr |- style="font-size:10pt" | height="15" | hold | 4C |} {| 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" | 10X NEBuffer 3.1 | align="center" align="center" | 1 | align="center" | 1x |} {| 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" | Temperature | width="72" align="center" valign="bottom" | Duration |- style="font-size:10pt" | height="15" | Digestion | 37C, 30min |- style="font-size:10pt" valign="bottom" | height="15" | heat inactivation | 80C, 20min |- style="font-size:10pt" | height="15" | 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=== {| 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" | |}
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