From:
`To:
`Subject:
`Date:
`Attachments:
`
`Shujun Luo
`John West
`Re: Can you please email me a copy of your slides from this afternoon?
`Thursday, November 1, 2012 4:26:21 PM
`labupdate121101.pptx
`
`Here it is.
`
`Thanks,
`
`Shujun
`
`On 11/1/12 4:00 PM, "John West" <john.west@personalis.com> wrote:
`
`>Thanks,
`
`Cell (408) 836-5586
`>john.west@personalis.com
`>
`
`>>
`
`>>
`
`John West
`
`Personalis EX2102
`
`
`To:
`Subject:
`Date:
`Attachments:
`
`Shujun Luo
`John West
`Re: Can you please email me a copy of your slides from this afternoon?
`Thursday, November 1, 2012 4:26:21 PM
`labupdate121101.pptx
`
`Here it is.
`
`Thanks,
`
`Shujun
`
`On 11/1/12 4:00 PM, "John West" <john.west@personalis.com> wrote:
`
`>Thanks,
`
`Cell (408) 836-5586
`>john.west@personalis.com
`>
`
`>>
`
`>>
`
`John West
`
`Personalis EX2102
`
`
`
`Exome Plus Lab Update
`
`Shujun Luo
`
`11/01/12
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`Personalis EX2102
`
`
`
`Personalis Exome Plus
`
`1. Standard Exome
`2. ESP
`3. LRP
`4. HGCP
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`2
`
`Personalis EX2102
`
`
`
`Workflow
`
`Gel free PCR free genomic prep
`
`Exome pull down prep
`
`DNA
`
`shear
`
`End repair
`
`spri
`
`+dATP
`
`ligation
`
`spri
`
`spri
`
`PCR
`
`spri
`
`First Hyb
`
`washing
`
`Second hyb
`
`washing
`
`PCR
`
`spri
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`3
`
`Personalis EX2102
`
`
`
`Impacts on Lab Processes
`
`Short shearing time
`
`……
`
`Long shearing time
`
`……
`
`Short Insert Library
`
`Short Insert Library
`
`Short Insert Library
`
`Long Insert Library
`
`Exome
`
`ESP
`
`HGCP
`
`LRP
`
`2 library preps
`4 hybridizations
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`4
`
`Personalis EX2102
`
`
`
`Single shearing
`
`……
`
`Consolidation
`
`Short Insert Library
`
`Short Insert Library
`
`Long Insert Library
`
`Exome + ESP
`
`HGCP
`
`LRP
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`5
`
`Personalis EX2102
`
`
`
`Single shearing
`
`……
`
`Consolidation
`
`Short Insert Library
`
`Exome + ESP + HGCP
`
`Long Insert Library
`
`LRP
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`6
`
`Personalis EX2102
`
`
`
`Goals
`
`1. Develop a single protocol to generate two libraries with different insert
`lengths.
`2. Rescue high GC contents and minimize coverage cv for GC%>70%.
`“Golden protocol” to enable Personalis Exome Plus in one tube.
`3.
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`7
`
`Personalis EX2102
`
`
`
`Single protocol two libraries
`
`DNA
`
`shear
`
`End repair
`
`Spri
`
`+dATP
`
`ligation
`
`Spri
`
`Spri
`
`PCR
`
`Spri
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`8
`
`Personalis EX2102
`
`
`
`Shear
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`9
`
`Personalis EX2102
`
`
`
`Spri
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`10
`
`Personalis EX2102
`
`
`
`Example
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`11
`
`Personalis EX2102
`
`
`
`GC bias
`
`Model System: Rhodobacter Sphaeroides
`
`The Rhodobacter sphaeroides ATCC 17025 genome is 4.56 Million bp long
`
`Browser Chrom/Plasmid Name Length (bp)
`chr
`
`
`plasmid_pRSPA01
`
`plasmid_pRSPA02
`
`plasmid_pRSPA03
`
`plasmid_pRSPA04
`
`plasmid_pRSPA05
`
`
`GC Content (%) Gene Count
`3217726
`68.48
`877879
`67.69
`289489
`67.60
`121962
`69.36
`36198
`64.05
`13873
`58.93
`
`NCBI RefSeq Accession
`
`3181
`
`849
`
`278
`
`114
`
`32
`
`12
`
`NC_009428
`NC_009429
`NC_009430
`NC_009431
`NC_009432
`NC_009433
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`12
`
`Personalis EX2102
`
`
`
`From Gabor:
`PST-000190
`
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000191
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000192
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000193
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000194
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000195
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000196
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000197
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000198
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`PST-000199
`GC > 80% (3404 regions):
`GC < 60% (13783 regions):
`
`mean
`24.09
`32.59
`
`29.07
`41.08
`
`30.1
`42.53
`
`28.48
`40.45
`
`25.89
`37.88
`
`1.03
`41.81
`
`31.29
`44.34
`
`25.23
`48.41
`
`22.86
`45.2
`
`23.97
`34.34
`
`third quartile
`29
`38
`
`rhodobacter pcr free
`median
`first quartile
`25
`21
`33
`29
`
`%_bases_above_15
`96.3
`99.9
`
`Normalized mean
`24.4
`33.1
`
`Delta
`8.6
`
`35
`47
`
`36
`49
`
`34
`46
`
`31
`44
`
`3
`55
`
`37
`51
`
`34
`55
`
`32
`52
`
`29
`40
`
`rhodobacter kapa 10 cycle
`30
`25
`42
`36
`
`98.9
`100
`
`rhodobacter kapa + betaine 10 cycle
`31
`27
`99
`43
`38
`100
`
`rhodobacter kapa + DMSO 10 cycle
`29
`25
`41
`35
`
`99.1
`100
`
`rhodobacter kapa hifi 10 cycle
`27
`22
`38
`33
`
`95.1
`99.9
`
`rhodobacter kapa hifi daza-dGTP 10 cycle
`2
`1
`0
`44
`30
`93
`
`rhodobacter kapa GC 10 cycle
`32
`27
`45
`39
`
`rhodobacter pmm 10 cycle
`28
`19
`48
`42
`
`99.1
`100
`
`80.8
`100
`
`rhodobacter pmm long denature 10 cycle
`25
`16
`76
`45
`39
`100
`
`rhodobacter kapa 8 cycle
`25
`21
`35
`30
`
`96.5
`99.9
`
`24.0
`33.9
`
`24.1
`34.1
`
`24.1
`34.2
`
`23.5
`34.3
`
`1.4
`55.2
`
`24.1
`34.1
`
`18.7
`35.9
`
`18.2
`36.0
`
`23.7
`33.9
`
`9.9
`
`10.0
`
`10.1
`
`10.9
`
`53.9
`
`10.0
`
`17.2
`
`17.8
`
`10.2
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`13
`
`Personalis EX2102
`
`
`
`H
`
`PO
`
`P
`OH
`
`-OH
`
`PA
`
`A
`T
`
`P
`
`OH-A
`
`T
`A
`
`Library Construction
`
`DNA
`
`shear
`
`End repair
`
`Spri
`
`+dATP
`
`ligation
`
`Spri
`
`Spri
`
`PCR
`
`Spri
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`14
`
`Personalis EX2102
`
`
`
`Areas to look into
`
`1. End Repair
`2. Shearing
`
`© Personalis, Inc. All Rights Reserved. COMPANY CONFIDENTIAL.
`
`15
`
`Personalis EX2102
`
`
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