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==Planning== * Sequential MDA on C1 with PEO1 * July 2, 2015 * Experiment ID: AR150702_C1 {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''(bases)''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | haploid length|| 3,100,000,000 |||||| |- | diploid length|| 6,200,000,000 |||||| |- | denature length|| 12,400,000,000 |||||| |- | |||||||| |- | phi29 mean processivity|| 70,000 |||||| |- | N9 combinations|| 262,144 ||N6|||| |- | % annealed template at 30 C||98%||2%|||| |- | |||||||| |- | desired gain||100||1000|||| |- | space between primers|| 691 || 61 |||| |- | # N9s to cover every base|| 1,377,777,778 |||||| |- | # N9s for spacing|| 1,968,254 |||||| |- | mols of N9 desired||3.27E-18|||||| |- | |||||||| |- | volume of first MDA (nL)|| 31.5 |||||| |- | volume of first MDA (L)||3.15E-08|||||| |- | concentration of N9 (M)||1.038E-10|||||| |- | concentration of N9 (nM)|| 0.1038 ||1|||| |- | fold change vs 50 uM|| 481,721 |||||| |- | |||||||| |- | primers (uM)||50|||||| |- | primers (M)||0.00005|||||| |- | |||||||| |- | ||C1 31 nL||Microwell 12 nL||volume (L)||1.20E-08 |- | dNTPs (mM)||4.00E-01|||||| |- | dNTPs (M)||4.00E-04|||||| |- | mol dNTPs||1.26E-11|||||| |- | # dNTPs||7.59E+12|||||| |- | mass/dNTPS incorporated (g/mol)||3.30E+02|||||| |- | mass of dntps (g)||4.16E-09|||||| |- | mass of dntps (pg)||4.16E+03|||||| |- | mass of single genome (pg)||6.6|||||| |- | gain|| 630 || 240 |||| |- | |} ===Protocol=== ====C1 setup==== [[File:AR150702_reagents-page-001.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-001.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-002.jpg|600px]] [[File:Protocol Worksheet AR150702_C1 v1-page-003.jpg|600px]] [[File:AR150702_thermalProfile.jpg|600px]] ====Off chip==== Prep * Add 1.2 uL 50X SYBR Green I to 120 uL NPM Nextera XT *Same as standard protocol, but add 15.5 uL NPM/SYBR Green I mix and run on qPCR machine (final volume 50.5 uL) 0.1X Tn5-059 *5 uL total **3 ul template **1 ul SSB **1 ul 1X Tn5-059 *Incubate 55 C for 5 min, transfer to cold block *Add 1 ul 0.2 mg/ml Qiagen Protease *Incubate 50 C for 15 min, followed by heat inactivation at 70 C for 20 min *Add 19 uL H2O to bring volume up from 6 uL to 25 uL *Add 5 uL each index (N7xx/S5xx) *Add 15.5 uL NPM/SYBR Green I mix qPCR protocol *72 C for 3 min *95 C for 1 min *25 cycles (but prepare to stop early) **95 C for 15 sec **55 C for 30 sec **72 C for 30 sec, acquisition **72 C for 15 sec *72 C for 5 min ==Results== ===C1 capture=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber''' | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Number''' | align="center" style="background:#f0f0f0;"|'''LiveOrDead''' | align="center" style="background:#f0f0f0;"|'''Notes''' |- | Ch01||A03||0||na||na |- | Ch02||A02||0||na||na |- | Ch13||C03||1||both||na |- | Ch04||A09||0||na||na |- | Ch05||A08||0||na||na |- | Ch06||A07||0||na||na |- | Ch07||B03||0||na||na |- | Ch08||B02||0||na||na |- | Ch14||C02||1||both||na |- | Ch10||B09||0||na||na |- | Ch11||B08||0||na||na |- | Ch12||B07||0||na||na |- | Ch18||C07||1||both||na |- | Ch24||D07||1||both||na |- | Ch15||C01||0||na||na |- | Ch16||C09||0||na||na |- | Ch25||E01||1||both||na |- | Ch26||E02||1||both||na |- | Ch19||D03||0||na||na |- | Ch20||D02||0||na||na |- | Ch29||E08||1||both||na |- | Ch30||E09||1||both||na |- | Ch23||Ch||0||na||na |- | Ch31||F01||1||both||na |- | Ch34||F07||3||both||na |- | Ch35||F08||1||both||na |- | Ch27||E03||0||na||na |- | Ch28||E07||0||na||na |- | Ch36||F09||1||both||na |- | Ch40||G07||1||both||na |- | Ch45||H03||1||both||extravasating |- | Ch32||F02||0||na||na |- | Ch33||F03||0||na||na |- | Ch48||H09||1||both||extravasating |- | Ch17||C08||2||dead||na |- | Ch21||D01||1||dead||na |- | Ch37||G01||1||dead||na |- | Ch38||G02||0||na||na |- | Ch39||G03||0||na||na |- | Ch44||H02||1||dead||na |- | Ch46||H07||1||dead||na |- | Ch42||G09||0||na||na |- | Ch43||H01||0||na||na |- | Ch47||H08||1||dead||na |- | Ch03||A01||1||live||na |- | Ch09||B01||1||live||na |- | Ch22||D09||1||live||weak |- | Ch41||G08||1||live||na |- | Ch49||A04||0||na||na |- | Ch50||A05||0||na||na |- | Ch51||A06||0||na||na |- | Ch52||A10||0||na||na |- | Ch53||A11||0||na||na |- | Ch54||A12||0||na||na |- | Ch55||B04||0||na||na |- | Ch56||B05||0||na||na |- | Ch57||B06||0||na||na |- | Ch58||B10||0||na||na |- | Ch59||B11||0||na||na |- | Ch60||B12||0||na||na |- | Ch61||C04||0||na||na |- | Ch62||C05||0||na||na |- | Ch63||C06||0||na||na |- | Ch64||C10||0||na||na |- | Ch65||C11||0||na||na |- | Ch66||C12||0||na||na |- | Ch67||D04||0||na||na |- | Ch68||D05||0||na||na |- | Ch69||D06||0||na||na |- | Ch70||D10||0||na||na |- | Ch71||D11||0||na||na |- | Ch72||D12||0||na||na |- | Ch73||E06||0||na||na |- | Ch74||E05||0||na||na |- | Ch75||E04||0||na||na |- | Ch76||E12||0||na||na |- | Ch77||E11||0||na||na |- | Ch78||E10||0||na||na |- | Ch79||F06||0||na||na |- | Ch80||F05||0||na||na |- | Ch81||F04||0||na||na |- | Ch82||F12||0||na||na |- | Ch83||F11||0||na||na |- | Ch84||F10||0||na||na |- | Ch85||G06||0||na||na |- | Ch86||G05||0||na||na |- | Ch87||G04||0||na||na |- | Ch88||G12||0||na||na |- | Ch89||G11||0||na||na |- | Ch90||G10||0||na||na |- | Ch91||H06||0||na||na |- | Ch92||H05||0||na||na |- | Ch93||H04||0||na||na |- | Ch94||H12||0||na||na |- | Ch95||H11||0||na||na |- | Ch96||H10||0||na||debris (clog) |- | |} ===Gels=== [[File:AR150702_C1_gelsSlide1.png|600px]] [[File:AR150702_C1_gelsSlide2.png|600px]] [[File:AR150702_C1_gelsSlide3.png|600px]] [[File:AR150702_C1_gelsSlide4.png|600px]] ===Sequencing=== First round of sequencing {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Index Sequence''' | align="center" style="background:#f0f0f0;"|'''# of Reads''' | align="center" style="background:#f0f0f0;"|'''Run Type, Minmum BP''' | align="center" style="background:#f0f0f0;"|'''Library Size''' | align="center" style="background:#f0f0f0;"|'''Read 1 Sequencing Primer''' | align="center" style="background:#f0f0f0;"|'''Index Primer''' | align="center" style="background:#f0f0f0;"|'''Read 2 Sequencing Primer''' | align="center" style="background:#f0f0f0;"|'''Notes''' |- | INITIAL-PROJECT-SAMPLE-DATE-Index||CGTGCA||Million||SE/PE, 36+6+36||SIZE RANGE (BP)||Name||Name||Name|| |- | AR-150702-D01-NxtXT-N702-S502||CGTACTAG-CTCTCTAT||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-E01-NxtXT-N702-S503||CGTACTAG-TATCCTCT||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-G01-NxtXT-N702-S505||CGTACTAG-GTAAGGAG||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-D01-CoRE-NEBNext25||TACTGC||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-E01-CoRE-NEBNext26||TCGTGA||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-G01-CoRE-NEBNext27||TACGCG||1.5||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-D01-Tn5059-N701-S506||TAAGGCGA-ACTGCATA||3||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 New |- | AR-150702-E01-Tn5059-N701-S507||TAAGGCGA-AAGGAGTA||3||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 New |- | AR-150702-G01-Tn5059-N701-S508||TAAGGCGA-CTAAGCCT||3||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 New |- | |} 2nd Round of sequencing {| {{table}} | align="center" style="background:#f0f0f0;"|'''SampleID''' | align="center" style="background:#f0f0f0;"|'''Index Sequence''' | align="center" style="background:#f0f0f0;"|'''# of Reads (M)''' | align="center" style="background:#f0f0f0;"|'''Run Type, Minmum BP''' | align="center" style="background:#f0f0f0;"|'''Size range (bp)''' | align="center" style="background:#f0f0f0;"|'''Read 1 Primer''' | align="center" style="background:#f0f0f0;"|'''Index 1 Primer''' | align="center" style="background:#f0f0f0;"|'''Read 2 Primer''' | align="center" style="background:#f0f0f0;"|'''Tube labeled''' |- | AR-150702-Ch03-A01-N703-S502||AGGCAGAA-CTCTCTAT||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool1 |- | AR-150702-Ch09-B01-N703-S503||AGGCAGAA-TATCCTCT||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool1 |- | AR-150702-Ch22-D09-N703-S505||AGGCAGAA-GTAAGGAG||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool1 |- | AR-150702-Ch44-H02-N703-S506||AGGCAGAA-ACTGCATA||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool1 |- | AR-150702-Ch45-H03-N703-S507||AGGCAGAA-AAGGAGTA||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool2 |- | AR-150702-Ch46-H07-N703-S508||AGGCAGAA-CTAAGCCT||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool2 |- | AR-150702-Ch47-H08-N703-S504||AGGCAGAA-AGAGTAGA||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool2 |- | AR-150702-Ch48-H09-N703-S517||AGGCAGAA-GCGTAAGA||2||SE50+8+8||200-800||HP10||HP12||NA||AR150714_Pool2 |- | AR-150702-D01-NxtXT-N702-S502||CGTACTAG-CTCTCTAT||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-E01-NxtXT-N702-S503||CGTACTAG-TATCCTCT||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-G01-NxtXT-N702-S505||CGTACTAG-GTAAGGAG||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-D01-CoRE-NEBNext25||TACTGC||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-E01-CoRE-NEBNext26||TCGTGA||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | AR-150702-G01-CoRE-NEBNext27||TACGCG||0.67||SE50+8+8||200-800||HP10||HP12||NA||DT Tn5 Old |- | |} ===Screening=== [[File:AR150702_libraryComplexities.jpg|600px]] ===CNV profiling=== [[File:AR-150702-Ch02ntc-A02-N702-S502.all.png|600px]] [[File:AR-150702-Ch03wklve-A01-N703-S502.all.png|600px]] [[File:AR-150702-Ch09wklve-B01-N703-S503.all.png|600px]] [[File:AR-150702-Ch21dea-D01-N702-S502.all.png|600px]] [[File:AR-150702-Ch22wklve-D09-N703-S505.all.png|600px]] [[File:AR-150702-Ch25bth-E01-N702-S503.all.png|600px]] [[File:AR-150702-Ch35bth-F08-N706-S517.all.png|600px]] [[File:AR-150702-Ch37dea-G01-N702-S505.all.png|600px]] [[File:AR-150702-Ch44dea-H02-N703-S506.all.png|600px]] [[File:AR-150702-Ch45lve-H03-N703-S507.all.png|600px]] [[File:AR-150702-Ch46dea-H07-N703-S508.all.png|600px]] [[File:AR-150702-Ch47dea-H08-N703-S504.all.png|600px]] [[File:AR-150702-Ch48bth-H09-N703-S517.all.png|600px]] [[File:DT-150701-bulk-PEO1-N704-S502.all.png|600px]] [[File:DT-150701-bulk-PEO4-N703-S517.all.png|600px]] ===MAPD profiling===
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