1/44
`
`Extract and isolate cell free polymicleotides
`from badily fluid
`
`102
`
`i fag double stranded polynucicotides with duplex
`tags (“Watson”strand tag distinguishable from
`“Crick” strand tag)
`
`Generate. sequence reads for tagged
`polynucleotide strands
`
`
`
`Reduce and/or track redundancy of reads to
`correspond to original strands
`
`Mapreads to selected locus or loci in genome
`
`
`
`164
`
`106
`
`108
`
`110
`
`Group reads into “Pairs” and “Singicts’”’:
`-Pairs are reads from complementary strands of
`original double stranded polynucleotides
`-- Singlets are reads for which there is no
`sequence read from the complementary strand of|\
`the original double stranded molecule
`\aq9
`
`100
`
`Determine quantitative measure of Pairs and
`Singlets mapping to each selected locus (also
`optionally read depth of paired and unpaired
`strands)
`
`
`
`
`
`
`
`
`Estimate number of Unseen polynucicotides in
`original population mapping to each locus
`
`
`Estimate number of Total polynucleotides in
`original population mapping to each locus
`
`
`
`Estimate Copy Member Variation at cach locus
`
`
`
`Fig. I
`
`\ 44
`
`1ié
`
`Lis
`
`120
`
`

`

`2/14
`
`2290 Total
`
`LocusB
`
`LocusA
`
`1440 pairs
`
`720 singlets 90 unseen
`
`
`
`
`
`sued QOOOL
`
`sjaibuls o9O, Ueesun 0gZ
`
`IE}OLOGE?
`
`Fig, 2
`
`

`

`3/711
`
`Locus A
`
`I i|
`
`reference
`
`sequence
`
`

`

`LocusA
`
`4/11
`
`Fig.4A
`
`amplification
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`ancBoBre
`
`
`
`Fig.4B
`
`oR
`
`wmyBR
`
`
`
`oO
`
`5
`
`3
`
`x
`
`S/11
`
`
`
`SequenceReads
`
`

`

`
`
` a,Lewimy'iLnvowmya:aarrrasxmzxmznnnrrrx'momg!gdiopfa6naaaa:eaeeeenerenelseen a¢5ca:necanCEO atas6xmz
`
`SequenceReads
`
`dD‘rea:
`eB
`
`
`
`
`
`te ww
`
`om ua
`
`ui
`
`12 wo
`
`1 or Gt OF
`
`
`
`
`
`-seeneeremmmmmenertaelb3nva, booObnqd—bnvd REREAD xg2xmzerence Correction1Lnyowmy1aMewgif¥anicSUAIEEE.oaanneanhaggLanCc—A——-B-----Aeeeenneeeeenesane
`
`
`
`
`
`xmzxmZz
`Complement
`Fig.4C
`ConsensusSequence
`
`
`
`
`
`
`fost ped Bagel
`
`3
`
`5!
`
`anc
`
`anc<aaeeennnn=nanannnnnmnnnnnnnnnsnneeakbna
`
`xmz
`
`Pair
`
`xmZz'5!
`
`6/11
`
`Collapseto
`
`5
`
`Wwm'¥3)
`|
`WwWvty
`
`

`

`fph
`
`
`
`
`
`
`
`€pg
`
`
`
`bVvd
`
`56ngg3—,moeabvdee_+aiadb'a
` aVvcsennaenancJanc3°55!epg“aepg'JeteeBCe.—1itwaeiitii€p8€Ppq€peGeFY_&Pe_955
`
`
`
`frsmonerarenpronnnnnnsCy1.'it2——on.naafncancNYafnc
`bndtT4,,t,itOH
`ondYe_aee3anYn6ad
`
`
`amplificationsequencingreduction
`vengenBo.EneEeDn
`€p9Sepg2™Nf0n
`;anca*fc5!¢3
`
`
`
`anc
`
`7/1
`
`redundancy
`
`

`

`8/11
`
`
`
`Fig. 6
`
`

`

`Podertoconmecttointeme
`Handhelldevicetoprovide
`
`sequencinginfarmationto
`
`
`z14OTTERSELTIPEEEELTDDi
`gfOERORRCRLEDIE,
`
`
`forerreerrsnserreernaccenttoanaenramanansnrrs
`
`
`
`
`#
`
`wet Og,
`
`2
`
`Cemputersystern
`
`ce
`
`reeeecerrnspoproreenns:
`neaeeeeonenss
`
`||
`
`
`
`9/11
`
`QUENCH
`
`if.
`
`seeerenereecvwsvrcenvecvoitapereeccoviin
`
`iek
`
`ee? OR BRR.
`
`gs. BE
`
`e#
`
`e#z
`
`4,
`
`Soo MEARE
`
`rereuser
`
`Fig.7
`
`
`
`
`
`
`
`

`

`10/44
`
`Provide cell free polynucleotides from bedily
`fluid of (a) a test sarnple and (b)} a control
`sarople
`
`Tag polynucleotides. Tag includes a source
`identifier (test or contro) and, optionally, a
`further identifier (optionally with duplex tags).
`Polynucicotides can be uniquely or non-uniquely
`tagged
`
`Pool tagged test and taggedcontrol
`polynucleotides at desired ratio (usually greater
`amount oftest)
`
`SOO
`
`Generate sequence reads from pooled test and
`control polynucleatides
`
`Reduce and/or track redundancy of reads to
`correspond to original molecules
`
`
`
`804
`
`7 806
`
`808
`
`810
`
`Quantify original molecules from each of test
`and control mapping te one or more genetic loci.
`(Can include normalizing against a reference
`locus}
`
`a function of test:control comparison
`
`Estimate copy number variation at each locus as
`y
`
`‘ig. 8
`
`

`

`14/14
`
`< §0 nucleotides
`
`. a
`
`Us
`
`*
`
`:
`
`3’
`
`Partial
`adaptor
`
`Lg
`
`Fig. 9A
`
`Fig. 9B
`
`Primer hybridization site
`
`Barcode
`
`sequence
`
`< 16 nucleotides
`
`Me
`
`rarAa
`
`Tagged
`polynucleotide
`
`Fig. 9C SFr
`— ‘SS
`&
`“
`
`Sample identification motif
`Se
`
`Sample identification motif f
`
`
`Sequencer motit
`
`Sequenecr motit
`
`

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