Andrew:Notebook/AR150702 C1: Difference between revisions

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==Sequential MDA on C1 with PEO1==
==Planning==
 
* Sequential MDA on C1 with PEO1
* July 2, 2015
* July 2, 2015
* Experiment ID: AR150702_C1
* Experiment ID: AR150702_C1


===Results===
{| {{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


====Gels====
*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_gelsSlide1.png|600px]]
Line 15: Line 337:
[[File:AR150702_C1_gelsSlide4.png|600px]]
[[File:AR150702_C1_gelsSlide4.png|600px]]


====Sequencing====
===Sequencing===


First round of sequencing
First round of sequencing
Line 96: Line 418:
|  
|  
|}
|}
===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===

Latest revision as of 20:41, 10 September 2015

Planning[edit]

  • Sequential MDA on C1 with PEO1
  • July 2, 2015
  • Experiment ID: AR150702_C1
' (bases) ' ' '
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[edit]

C1 setup[edit]

File:AR150702 reagents-page-001.jpg File:Protocol Worksheet AR150702 C1 v1-page-001.jpg File:Protocol Worksheet AR150702 C1 v1-page-002.jpg File:Protocol Worksheet AR150702 C1 v1-page-003.jpg File:AR150702 thermalProfile.jpg

Off chip[edit]

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[edit]

C1 capture[edit]

Chamber Well Number LiveOrDead 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[edit]

File:AR150702 C1 gelsSlide1.png

File:AR150702 C1 gelsSlide2.png

File:AR150702 C1 gelsSlide3.png

File:AR150702 C1 gelsSlide4.png

Sequencing[edit]

First round of sequencing

Sample ID Index Sequence # of Reads Run Type, Minmum BP Library Size Read 1 Sequencing Primer Index Primer Read 2 Sequencing Primer 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

SampleID Index Sequence # of Reads (M) Run Type, Minmum BP Size range (bp) Read 1 Primer Index 1 Primer Read 2 Primer 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[edit]

File:AR150702 libraryComplexities.jpg

CNV profiling[edit]

File:AR-150702-Ch02ntc-A02-N702-S502.all.png File:AR-150702-Ch03wklve-A01-N703-S502.all.png File:AR-150702-Ch09wklve-B01-N703-S503.all.png File:AR-150702-Ch21dea-D01-N702-S502.all.png File:AR-150702-Ch22wklve-D09-N703-S505.all.png File:AR-150702-Ch25bth-E01-N702-S503.all.png File:AR-150702-Ch35bth-F08-N706-S517.all.png File:AR-150702-Ch37dea-G01-N702-S505.all.png File:AR-150702-Ch44dea-H02-N703-S506.all.png File:AR-150702-Ch45lve-H03-N703-S507.all.png File:AR-150702-Ch46dea-H07-N703-S508.all.png File:AR-150702-Ch47dea-H08-N703-S504.all.png File:AR-150702-Ch48bth-H09-N703-S517.all.png File:DT-150701-bulk-PEO1-N704-S502.all.png File:DT-150701-bulk-PEO4-N703-S517.all.png

MAPD profiling[edit]