Case 1:20-cv-01580-LPS Document 42-1 Filed 03/05/21 Page 1 of 451 PageID #: 9440
`Case 1:20-cv-01580-LPS Document 42-1 Filed 03/05/21 Page 1 of 451 PageID #: 9440
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`EXHIBIT 125
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`EXHIBIT 125
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`

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`Case 1:20-cv-01580-LPS Document 42-1 Filed 03/05/21 Page 2 of 451 PageID #: 9441
`
`US010752951B2
`
`( 12 ) United States Patent
`Salk et al .
`
`US 10,752,951 B2
`( 10 ) Patent No .:
`( 45 ) Date of Patent :
`* Aug . 25 , 2020
`
`( 54 )
`
`METHODS OF LOWERING THE ERROR
`RATE OF MASSIVELY PARALLEL DNA
`SEQUENCING USING DUPLEX CONSENSUS
`SEQUENCING
`( 71 ) Applicant : UNIVERSITY OF WASHINGTON
`THROUGH ITS CENTER FOR
`COMMERCIALIZATION , Seattle ,
`WA ( US )
`( 72 ) Inventors : Jesse Salk , Seattle , WA ( US ) ;
`Lawrence A. Loeb , Bellevue , WA
`( US ) ; Michael Schmitt , Seattle , WA
`( US )
`( 73 ) Assignee : UNIVERSITY OF WASHINGTON
`THROUGH ITS CENTER FOR
`COMMERCIALIZATION , Seattle ,
`WA ( US )
`Subject to any disclaimer , the term of this
`patent is extended or adjusted under 35
`U.S.C. 154 ( b ) by 0 days .
`This patent is subject to a terminal dis
`claimer .
`( 21 ) Appl . No .: 16 / 514,931
`( 22 ) Filed :
`Jul . 17 , 2019
`( 65 )
`Prior Publication Data
`Nov. 7 , 2019
`US 2019/0338358 A1
`
`( * ) Notice :
`
`( 60 )
`
`Related U.S. Application Data
`Division of application No. 16 / 120,091 , filed on Aug.
`31 , 2018 , now Pat . No. 10,370,713 , which is a
`continuation of application No. 15 / 660,785 , filed on
`Jul . 26 , 2017 , now Pat . No. 10,287,631 , which is a
`continuation of application No. 14 / 386,800 , filed as
`application No. PCT / US2013 / 032665 on Mar. 15 ,
`2013 , now Pat . No. 9,752,188 .
`
`( 51 )
`
`( 60 ) Provisional application No. 61 / 613,413 , filed on Mar.
`20 , 2012 , provisional application No. 61 / 625,623 ,
`filed on Apr. 17 , 2012 , provisional application No.
`61 / 625,319 , filed on Apr. 17 , 2012 .
`Int . CI .
`C12Q 1/68
`( 2018.01 )
`( 2018.01 )
`C12Q 1/6876
`( 2018.01 )
`C12Q 1/6806
`( 2018.01 )
`C12Q 1/6869
`U.S. CI .
`CPC
`C12Q 1/6876 ( 2013.01 ) ; C12Q 1/6806
`( 2013.01 ) ; C12Q 1/6869 ( 2013.01 )
`Field of Classification Search
`C12Q 1/6806 ; C12Q 1/6869 ; C12Q
`CPC
`2525/191 ; C12Q 2535/119 ; C12Q
`2563/179
`See application file for complete search history .
`
`( 52 )
`
`( 58 )
`
`( 56 )
`
`References Cited
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`
`Primary Examiner - David C Thomas
`( 74 ) Attorney , Agent , or Firm - Perkins Coie LLP ; Lara
`J. Dueppen
`
`( 57 )
`ABSTRACT
`Next Generation DNA sequencing promises to revolutionize
`clinical medicine and basic research . However , while this
`technology has the capacity to generate hundreds of billions
`of nucleotides of DNA sequence in a single experiment , the
`error rate of approximately 1 % results in hundreds of
`millions of sequencing mistakes . These scattered errors can
`be tolerated in some applications but become extremely
`problematic when " deep sequencing " genetically heteroge
`neous mixtures , such as tumors or mixed microbial popu
`lations . To overcome limitations in sequencing accuracy , a
`method Duplex Consensus Sequencing ( DCS ) is provided .
`This approach greatly reduces errors by independently tag
`ging and sequencing each of the two strands of a DNA
`duplex . As the two strands are complementary , true muta
`tions are found at the same position in both strands . In
`contrast , PCR or sequencing errors will result in errors in
`only one strand . This method uniquely capitalizes on the
`redundant information stored in double - stranded DNA , thus
`overcoming technical limitations of prior methods utilizing
`data from only one of the two strands .
`
`28 Claims , 12 Drawing Sheets
`Specification includes a Sequence Listing .
`
`A2190
`
`

`

`Case 1:20-cv-01580-LPS Document 42-1 Filed 03/05/21 Page 3 of 451 PageID #: 9442
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`References Cited
`OTHER PUBLICATIONS
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`* cited by examiner
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`Sheet 1 of 12
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`Figure 1
`
`SMI sequences
`T - tailed DNA fragment
`
`***
`
`Ligation &
`size selection
`
`adaptor sequence
`
`CB SM family
`
`WS
`
`Ba SM family
`0.00
`
`********
`
`Capture target regions
`
`Massively parallel sequencing
`
`A2195
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`Figure 2
`
`***
`
`*** ...
`
`*** ***** ::
`
`:
`
`444 NHNNNNNNNNNGCA
`
`SEQ ID NO : ng
`
`?
`
`*****
`***
`
`*** *****
`
`NNNNNNNN SAGA
`
`SEQ ID NO : 8
`SEQ ID NO : 7
`
`polymerase CATP
`
`NNNNNNNS ASIA SEQ ID NO : 7
`
`SEQ ID NO : 9
`
`A2196
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`Sheet 3 of 12
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`Figure 3
`
`b
`
`..........
`
`*
`
`Error - corrected
`consensus sequences
`
`Mutation counting among
`consensus sequences
`
`O Wild - type : 7/10
`Mutation prevalence : 30 %
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`A2197
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`Figure 4
`
`A
`
`5 '
`3 '
`
`1 - TAAC
`2 - ATTG
`
`-TCCG - 2
`--AGGC - 1
`
`3 '
`5 '
`
`( top strand )
`( bottom strand )
`
`B
`
`C
`
`5 '
`
`5 '
`
`1 - TAAC
`
`1 - CGGA-
`
`-TCCG - 2
`
`-GTTA - 2
`
`3 '
`
`3 '
`
`( top strand )
`
`( bottom strand )
`
`1 - TAAC ----
`2 - ATTG -------
`
`( top strand )
`TCCG - 2
`-AGGC - 1 ( bottom strand ) )
`
`A2198
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`Sheet 5 of 12
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`
`
` p < 1e - 15
`
`
`E
`
`
`
` p < 1e - 15
`
`[
`
`]
`
`
`
` p < 1e - 15
`
`
`
`
`
`SSCS analysis
`
`5e - 4
`
`4e - 4
`
`3e - 4
`
`2e - 4
`
`1e - 4
`
`0
`
`
`
`Mutation frequency
`
`D
`
`1.0e - 2
`
`
`
`Q30 analysis
`
`A
`
`
`
`log scale
`
`1.0e - 3
`
`2.00-4
`
`Mutation frequency
`
`1.5e - 4
`
`1.0e - 4
`
`5.0e - 5
`
`DCS
`
`SSCS
`
`
`Q30 reads
`A
`
`Frequency 0.02
`0.04
`0.06
`B
`
`Mutation
`
`Figure 5
`
`
`
`DSC analysis
`
`Type of mutation
`
`Transversions
`AGI- > CG - ACTGCCGACT - GA - TT- > AG - TC A
`0076
`Transitions
`
`Transversions
`ol 1e - 5
`
`Type of mutation
`
`
`
`A GT - CG - ACTGCCGA- > CT - GATTA GTCA
`
`Transitions
`
`0
`
`4e - 5
`
`3e - 5
`
`2e - 5
`
`
`
`Mutation frequency
`E
`
`
`
`DCS analysis
`
`15000
`10000 Genome position 5000
`
`
`
`15000
`
`
`10000 Genome position 5000
`
`
`0.00 0
`0.02 ]
`0.06
`
`Mutation Frequency
`C
`
`0.04
`
`0.001 0
`
`A2199
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`Sheet 6 of 12
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`Figure 6
`
`Consensus Sequencing removes artifactual sequencing errors
`
`4.0e - 3
`
`3.5e - 3
`
`7e - 5
`
`6e - 5
`
`5e - 5
`
`
`
`50 - fold magnification 4e - 5
`
`3e - 5 *
`
`
`
`Mutation frequency 2.0e - 3
`
`3.0e - 3
`
`2.5e - 3
`
`1.5e - 3
`
`1.0e - 3
`
`0.5e - 3
`
`2e - 5
`
`1e - 5
`
`Reference Raw reads
`SSCS DCS
`
`Reference Raw reads
`SSCS DCS
`
`A2200
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`Sheet 7 of 12
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`Figure 7
`? 4e - 5
`
`3e - 5
`
`
`
`Mutation frequency 2e - 5
`
`1e - 5
`
`SSCS
`DCS
`Reference
`
`< 0.01
`Ipa
`
`] p < 0.01
`
`0
`AG , F > C G?A , C TG > C , C + GA > c , 32GA > T , T + A G > T , CPA
`Transversions
`Transitions
`Type of mutation
`
`that
`
`7e - 5
`
`6e - 5
`
`B
`
`p < 1e - 15
`
`
`
`Mutation frequency 4e - 5
`
`5e - 5
`
`3e - 5
`
`2e - 5
`
`1e - 5
`
`p < 1e - 15
`
`to 000
`
`??????
`
`A - GT- > CGA C - T G- C CG ACT - GATTA GT C- > A
`Transversions
`Transitions
`Type of mutation
`
`A2201
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