WP 2016-527313 A 2016.9.8
`
`(19) ASSESUP)
`
`ands Fe He FF 2 HCA)
`
`() SHEBARSS
`#92%2016-527313
`(P2016-527313A)
`(43) 220 S#R2849F 8A (2016.9. 8)
`
`C40B
`10/00
`C1l2N
`15/00
`C12P
`19/34
`GOIN
`37/00
`A
`9/16
`C12N
`BERR RAR Pie@Hiek APR (4 251 A)
`
`ZNA
`
`A
`
`101
`
`F-VA—-k (Bs)
`4B050
`4B064
`
`RRRRIC
`
`F 1
`
`(51) Int. Cl.
`C40B 10/00
`C12N_ 15/09
`C1i2P 19/34
`GOIN 37/00
`Ci2N YIé
`
`(2006. 07)
`(2006. 01)
`(2006. 01)
`(2006. 07)
`(2006. 01)
`
`2) Hes
`(86) (22) HEA
`(85) #aRD HE A
`(86) BR HRB
`87) BRAHBS
`(87) BR&SHA
`Gl) FXBEREE
`(32) 48
`33) RABEBH
`Ql) RHEERES
`(2) EAE
`(33) BEES
`
`49BB 2016-533384 (P2016-533384)
`5F 26478A 5 A (2014.8.5)
`SF AR28473318 (2016.3. 31)
`PCT/US2014/049834
`W02015/021080
`SP 2727 128 (2015. 2. 12)
`61/862, 445
`5F A254F8A 5 A (2013.8. 5)
`3k Bl (US)
`61/862, 457
`37 25478A 5 Bl (2013.8.5)
`38 El (US)
`
`(64) FRHOSE) PRAM LAMBERTI 17S)
`
` (74) (CHB A
`
`(71) HERA 516040017
`VFA AIAPTOIYA A-RE—
`vay
`VAUAGRH 94158 BWVFaW
`=TM YF YYAd
`ivray:
`4 Fok OA 455 A
`4—-p 545
`100082072
`Ft FR Re
`Ko-d, VAUTL
`FAUAGRH 94134 AVTFaWv
`=7TW YR YY Ad Vitae e
`-ARYU-b 738
`
`(72) FEARS
`
`FRATlt S
`
`67) (24)
`
`(EWLF—-BEAT SO, BROMMAKENEASE
`
`SA FFU MAME CHKENS. FHI, FYVAR
`
`DULAF FREDO, BRBORRIO yIOHEDKH
`
`DNTKAAMR, APMBIMMENS, FY RBA
`
`. VIFTORAP RY ATV ERALT. PA LOCARS
`
`NHS. SHI, AAMBOARS, RU. BHAOB
`
`GBEOKEGIT ID JOMUBREMEICT S. Rb
`BOR RBORBOKEES ST ISU OBREOELDONT
`N41 Ald, AAMBCE SITAR ENS,
`
`[2RE] B3
`
`Figure 3
`Oligo Printer
`
` edbdeelTtHe
`
`‘eile
`
`

`

`7/20/2020
`
`Espacenet- Bibliographic data
`
`JN2SHSosssyNyaysyaualaSsagi‘aere)=JR%Rasaoases=sll
`SogouaSaoosaitstSoy,1='.G3OO)nellSESECo}5]catSecalSeealcdeicaPOSieyist&2“ne&§gySNas-BeoDA£§2a)asse—y_-\25tO—:waVN+B85Go43)aeBegaXK=2=<itemmleeA®oOoOqaRAS
`
`
`ehxn|SHogSaastaaQ@ann\#Sasa;vemonqn&lSolBis)3]BlhesmiSiiDOS~Dies
`
`
` yoZz1:ceonyeawtsohOnac-©r%wnaz©oDomerr¢°Surc®62£8&$3232a====enoa82ESE85weeti>a.wyOo5ts£5Hadey=<{oOqeanc€a
`savasogaodoo©tia~\oOSUSaaTOSSISISOC4=el3agenN+MSei.aSaasOeSAmfoy:oNsniSormFHEEedoeAGtLemtet]ee,<8&oyyssooaeatgad\agcssagsoOwie
`
`
`
`waeweeiYWVvOrgpodangaSalomebatemladerbseedonehomeOSOeOi!
`oe:eeNaNSOEcoors2SSeaSISS=CHen)CaaCSCaeeCorLestBedoSonusyKSSAMOGAGGed:beHH&eC2)8CALCalilGlcalGxSQ}ulPtCOTLOSr]LLfed-~LtPLLCNTLust:oe=)ai=5Nic2afae“3otSSa)Ahma]da)dfmaMma)nogoaaanvwinanBAABs
`
`
`
`‘i||a.PoaTiaoOQ.tiwles5BGs
`ieddeddedceeie]Sl!ooSsBrin:fe,~¢btOO)Sicolaul=iwitH0.
`(OloewomaSUINGGhaaoaqgStns.ms
`
`:”eaSiIqae—eeKof=ge2OOtOoemyCO)a]LL°aated:eS=~4moa7Wege:~{ufoSaiiLfLutoOAeeeet]2AO)5SLOOLeg,aBoyvBRAWoSeenalo8OyiCea:a\i=iflSo.afoDeHik©’Saas
`
`aneZe“=NNeetonname
`free:aiAG)efCotNHel]ONTETNEcntNYoNoOfoe
`:|BmealooeyPS!OoLWNoNro)wd
`oye£OoOQSaaaonNXxXoNa|-_=)C31LE
`
`
`..oe=OHealSS)OD)colLO)colrhNcNeietoat
`
`\N|eugeaul3B==‘SSie-€FSaibe4osKHFasoSSoy2BaisXN4oyGHST)
`cayoy52)oo©os
`
`esl8“3uuNAOeOla)ATTOCTchmlotShee=
`'oeon.+SS~agGa.oyonaik=aoeSFBSalwitlAl
`zm(UMaageeeeeCOACO2efLed
`
`oO‘agraii3OlaeelodeetOHOOOY<A]OlOf|aC35BEF
`anSl
`
`https://worldwide.espacenet.com/publicationDetails/biblio?7DB
`
`EPODOC&II
`
`1&ND
`
`3&adjacent=true&locale:
`
`en_EP&FT=D&date
`
`20160908&CC
`
`=JP...
`
`1/2
`
`
`
`

`

`7/20/2020
`
`Espacenet- Bibliographic data
`
`https://worldwide.espacenet.com/publicationDetails/biblio?7DB
`
`EPODOC&IF1&ND
`
`=3&adjacent=true&locale=
`
`en_EP&FT=D&date
`
`20160908&CC=JP...
`
`2/2
`
`
`

`

`
`
`Notice
`This translation is machine-generated. It cannot be guaranteed thatit is intelligible, accurate,
`complete, reliable orfit for specific purposes. Critical decisions, such as commercially relevant or
`financial decisions, should not be based on machine-translation output.
`
`DESCRIPTION JP2016527313A
`70 Abstract Provided herein is a large newly synthesized library of nucleic acids that has a low
`error rate. Further described herein are devices for the production of high quality building
`blocks, such as oligonucleotides. Longer nucleic acids can be synthesized in parallel using
`microfluidic assembly. In addition, the methods herein allow for fast construction of large
`libraries of long, high quality genes. Devices for the production of large libraries of long and
`high quality nucleic acids are further described herein. [Selection diagram] Fig. 3
`16 Newly synthesized gene library
`
`[0001]
`20 CROSS REFERENCE This application claims the benefit of US Provisional Patent Application No.
`61/862,445 filed August 5, 2013 and US Provisional Patent Application No. 61/862,457 filed
`August 5, 2013. The application is incorporated herein by reference.
`
`[0002]
`26 Highly fidelity and low cost highly efficient chemical gene synthesis has a central role in
`biotechnology and medicine andin basic biological research.
`28 Novel gene synthesis is a powerful tool for basic biological research and applicationsin
`biotechnology. Various methods are knownfor the synthesis of relatively short fragments on a
`small scale, but these techniques suffer from scalability, automation, speed, accuracy, and cost.
`Whatis neededis a device for a simple, reproducible, scalable, less error prone, and cost-
`effective method that ensures successful synthesis of the desired gene and is applicable to
`automation. ing.
`
`[0003]
`37 AS noted above,there is a rapid need for methods, devices and systemsthat can rapidly
`
`21-07-2020
`
`1
`
`

`

`synthesize large genelibraries or relatively longer oligonucleotide fragments with less error
`and efficiency. Similarly, there is a need for methodsthat can partition and mix liquid reagents
`on a microfluidic scale for many individually addressable reactions in parallel. The present
`invention addresses these needsand also provides related advantages.
`
`[0004]
`45 In one aspect, the specification provides a genelibrary as described in the present invention. A
`genelibrary contains a collection of genes. In some embodiments, the assemblyis at least 100
`different preselected, which may be at least 0.5 kb in length with an error rate of less than 1
`at 3000 bp compared to a predetermined sequence comprising the gene. Included synthetic
`genes. In another aspect, the invention also provides a gene library comprising a collection of
`genes. The assembly mayinclude at least 100 different preselected synthetic genes, each of
`which may beat least 0.5 kb in length. At least 90% of the preselected synthetic genes may
`contain an errorrate of less than 1 in 3000 bp comparedto the predetermined sequence
`containing the gene. The desired predetermined sequence can be provided by any method,
`typically by the user, eg, the user who will enter the data using a computerized system. In
`various embodiments, the synthesized nucleic acids are, in some cases, predetermined by
`sequencingat least a portion of the synthesized nucleic acids, for example, using next
`generation sequencing methods. Is compared to the sequence. In some embodiments related
`to any of the gene libraries described herein, at least 90% of the preselected synthetic genes
`are 1 in 5000 bp compared to the predetermined sequencecontaining the gene. Including an
`error rate of less than. In some embodiments,at least 0.05% of the preselected synthetic genes
`are errorfree. In some embodiments,at least 0.5% of the preselected synthetic genes are error
`free. In some embodiments, at least 90% of the preselected synthetic genes contain an error
`rate of less than 1 in 3000 bp as compared to the predetermined sequence containing the
`gene. In some embodiments, at least 90% of the preselected synthetic genes are error-free or
`error-free. In some embodiments, the preselected synthetic gene comprises a deletion rate of
`less than 1 at 3000 bp as comparedto a predetermined sequence comprising the gene.In
`some embodiments, the preselected synthetic gene comprises an insertion rate of less than 1
`in 3000 bp as compared to a predetermined sequence comprising the gene. In some
`embodiments, the preselected synthetic gene comprises a substitution rate of less than 1 at
`3000 bp as compared to a predetermined sequence comprising the gene.
`In some embodiments, the genelibrary as described herein further comprises at least 10
`copies of each synthetic gene. In some embodiments, the genelibrary as described herein
`further comprises at least 100 copies of each synthetic gene. In some embodiments, the gene
`library as described herein further comprises at least 1000 copies of each synthetic gene.In
`some embodiments, the gene library as described herein further comprises at least 1,000,000
`copies of each synthetic gene. In some embodiments, the collection of genes as described
`herein comprises at least 500 genes. In some embodiments, the assembly comprises at least
`5000 genes. In some embodiments, the population comprises at least 10,000 genes. In some
`embodiments, the preselected synthetic geneis at least 1 kb. In some embodiments, the
`
`7 ~
`
`21-07-2020
`
`2
`
`

`

`preselected synthetic geneis at least 2 kb. In some embodiments, the preselected synthetic
`gene is at least 3 kb. In some embodiments, the predetermined sequence additionally
`comprises less than 20 bp as comparedto the preselected synthetic gene. In some
`embodiments, the predetermined sequenceadditionally comprises less than 15 bp as
`compared to the preselected synthetic gene. In some embodiments,at least one of the
`synthetic genes differs from other synthetic genes by at least 0.1%. In some embodiments,
`each synthetic gene differs from other synthetic genes by at least 0.1%. In some embodiments,
`at least one of the synthetic genes differs from other synthetic genes by at least 10%. In some
`embodiments, each synthetic gene differs from other synthetic genes by at least 10%. In some
`embodiments,at least one of the synthetic genes differs from other synthetic genes by at least
`two base pairs. In some embodiments, each synthetic gene differs from other synthetic genes
`by at least two base pairs. In some embodiments, the gene library as described herein further
`comprises a synthetic gene that is less than 2 kb with an errorrate of less than 1 at 20,000
`bp, as compared to a preselected sequence of genes. .. In some embodiments, the subset of
`deliverable genes are covalently linked together.
`In some embodiments,the first subset of the collection of genes encodes a componentof the
`first metabolic pathway with one or more metabolic end products. In some embodiments, a
`gene library as described herein further comprises the selection of one or more metabolic end
`products, thereby building a collection of genes. In some embodiments, the one or more
`metabolic end products comprises biofuels. In some embodiments, the second subsetof the
`collection of genes encodes components of the second metabolic pathway with one or more
`metabolic end products. In some embodiments, the gene library is in a space thatis less than
`100m<3>. In some embodiments, the gene library is in a space that is less than 1 m<3>. In
`some embodiments, the genelibrary is in a space thatis less than 1 m<3>.
`
`95
`
`[0005]
`107 In another aspect, the invention also provides a method of constructing a genelibrary. The
`method comprises the steps of inputting at least a first list of genes and a secondlist of
`genes into a computer readable non-transitory medium priorto a first time point, wherein
`the genesare at least 500 bp and compiled into a binding list. When the bindinglist
`comprises at least 100 genes, and synthesizing 90% or moreof the genesin the bindinglist
`before the second timepoint, thereby constructing a genelibrary with deliverable genes.
`including. In some embodiments, the second time point is less than one month apart from the
`first time point.
`
`[0006]
`718 In performing any of the methods of constructing a genelibrary as provided herein, the
`method as described herein further comprises delivering at least one gene at a second time
`point. In some embodiments, at least one of the genes differs from the other genebyatleast
`0.1% in the genelibrary. In some embodiments, each of the genes differs from the other
`
`21-07-2020
`
`3
`
`

`

`genesbyat least 0.1% in the gene library. In some embodiments, at least one of the genes
`differs from the other gene by at least 10% in the genelibrary. In some embodiments, each of
`the genesdiffers from the other genes by at least 10% in the genelibrary. In some
`embodiments,at least one of the genes differs from the other genebyat least two base pairs
`in the genelibrary. In some embodiments, each of the genes differs from the other gene by at
`least two base pairs in the genelibrary. In some embodiments,at least 90% of the deliverable
`genesare errorfree. In some embodiments, the deliverable gene comprises an error rate of
`less than 1/3000,resulting in a sequence that deviates from the sequence of the gene in the
`linked list of genes. In some embodiments,at least 90% of the deliverable genes have an
`error rate of less than 1 in 3000 bp, resulting in sequences that deviate from the sequences
`of the genesin thelinkedlist of genes. In some embodiments, the genesin the subset of
`deliverable genes are covalently linked together. In some embodiments,the first subset of the
`linked list of genes encodes a componentofthe first metabolic pathway with one or more
`metabolic end products. In some embodiments, any of the methods of constructing a gene
`library as described herein further comprises selecting one or more metabolic end products,
`wherebythefirst list of genes,the first list. 2 Build a list orjoin list. In some embodiments,
`the one or more metabolic end products comprises biofuels. In some embodiments, the
`second subsetof the linked list of genes encodes a componentof the second metabolic
`pathway with one or more metabolic end products. In some embodiments,the linked list of
`genes comprises at least 500 genes. In some embodiments, the linked list of genes comprises
`at least 5000 genes.
`743 IN some embodiments,the linked list of genes comprises at least 10,000 genes. In some
`embodiments, the gene can beat least 1 kb. In some embodiments, the geneis at least 2 kb.
`In some embodiments, the gene is at least 3 kb. In some embodiments, the second time point
`is less than 25 days away from the first time point. In some embodiments, the second time
`point is less than 5 days away from thefirst time point. In some embodiments, the second
`time pointis less than two days away from thefirst time point.It is noted that any of the
`embodiments described herein may be combinedwith any of the methods, devices or
`systems provided herein.
`
`[0007]
`154 In another aspect, methods of constructing a genelibrary are provided herein. The method
`comprises inputting a list of genes into a computer readable non-transitory medium at a first
`time point, synthesizing 90% or moreofthe list of genes, thereby building a genelibrary with
`deliverable genes. , And delivering the gene deliverable at the second time point. In some
`embodiments, the list comprises at least 100 genes and genes maybeat least 500 bp. Still
`further, in some embodiments, the second time pointis less than a month apart from the first
`time point.
`
`[0008]
`
`21-07-2020
`
`4
`
`

`

`164 In carrying out any of the methods of constructing a genelibrary as provided herein, in some
`embodiments, the method as described herein comprises at least one gene at a second time
`point. The method further comprises the step of delivering. In some embodiments, at least
`one of the genesdiffers from the other geneby at least 0.1% in the genelibrary. In some
`embodiments, each of the genes differs from the other genes by at least 0.1% in the gene
`library. In some embodiments,at least one of the genes differs from the other gene by at
`least 10% in the genelibrary. In some embodiments, each of the genes differs from the other
`genesbyat least 10% in the genelibrary. In some embodiments,at least one of the genes
`differs from the other gene by at least two base pairs in the genelibrary. In some
`embodiments, each of the genesdiffers from the other gene byat least two base pairs in the
`genelibrary. In some embodiments, at least 90% of the deliverable genesare errorfree. In
`some embodiments, the deliverable genes have an errorrate of less than 1/3000,resulting in
`sequencesthat deviate from the sequencesof the genesin thelist of genes. In some
`embodiments,at least 90% of the deliverable genes have an errorrate of less than 1 in 3000
`bp, resulting in sequences that deviate from the sequences of the genesin thelist of genes. In
`some embodiments, the genes in the subset of deliverable genes are covalently linked
`together. In some embodiments,the first subset of the list of genes encodes a componentof
`the first metabolic pathway with one or more metabolic end products. In some embodiments,
`the methodof constructing a genelibrary further comprises selecting one or more metabolic
`end products, thereby constructing a list of genes. In some embodiments, the one or more
`metabolic end products comprises biofuels. In some embodiments, the second subset of the
`list of genes encodes componentsof the second metabolic pathway with one or more
`metabolic end products.It is noted that any of the embodiments described herein may be
`combined with any of the methods, devices or systems provided herein.
`
`[0009]
`191 In practicing any of the methods of constructing a genelibrary as provided herein, in some
`embodiments,the list of genes comprises at least 500 genes. In some embodiments,the list
`comprises at least 5000 genes. In some embodiments, the list comprises at least 10,000
`genes. In some embodiments, the geneis at least 1 kb. In some embodiments, the geneis at
`least 2 kb. In some embodiments, the geneis at least 3 kb. In some embodiments, the second
`time pointis less than 25 days apart from thefirst time point as described in the method of
`constructing a genelibrary. In some embodiments, the second timepointis less than 5 days
`awayfrom thefirst time point. In some embodiments, the second timepointis less than two
`days away from thefirst time point.It is noted that any of the embodiments described herein
`may be combined with any of the methods, devices or systems provided herein.
`
`[0010]
`204 In another aspect, the invention also provides a method of synthesizing an n-mer
`oligonucleotide on a substrate. The method comprises a) providing to the substrate a
`
`21-07-2020
`
`5
`
`

`

`degradedloci functionalized with a chemical moiety suitable for nucleotide binding, and b) at
`least 12 per hour according to a location-specific predetermined sequence.At a rate of
`nucleotides of at least two building blocks are attached to a plurality of growing
`oligonucleotide chains, each of which is present at one of the degraded positions, whereby n
`base pair lengths are present. Synthesizing a plurality of oligonucleotides. Various
`embodimentsrelated to methods of synthesizing n-meroligonucleotides on a substrate are
`described herein.
`
`[0011]
`216 In any of the methodsof synthesizing an n-meroligonucleotide on a substrate as provided
`herein, in some embodiments, the method decomposeseachata rate of at least 15
`nucleotides per hour. The method further comprises attaching at least two building blocks to
`a plurality of growing oligonucleotide chains present at one of the locations. In some
`embodiments, the method attachesat least two building blocks at a rate of at least 20
`nucleotides per hour to a plurality of growing oligonucleotide chains, each present at one of
`the degraded sites. The method further includes a step. In some embodiments, the method
`binds at least two building blocksat a rate of at least 25 nucleotides per hour to a plurality of
`growing oligonucleotide chains, each present at one of the degraded sites. The method
`further includes a step. In some embodiments, the at least one degradedsite comprises an n-
`meroligonucleotide that deviates from a predetermined sequencespecific for a site with an
`error rate of less than 1/500 bp. In some embodiments,at least one degraded site comprises
`an n-mer oligonucleotide that deviates from a predetermined sequencespecific for a site with
`an error rate of less than 1/1000 bp. In some embodiments, at least one degraded site
`comprises an n-meroligonucleotide that deviates from a predetermined sequence specific for
`a site with an errorrate of less than 1/2000 bp. In some embodiments,the plurality of
`oligonucleotides on the substrate deviates from a predetermined sequencespecific for each
`location with an error rate of less than 1/500 bp. In some embodiments,the plurality of
`oligonucleotides on the substrate deviates from the predetermined sequencespecific for each
`location with an errorrate of less than 1/1000 bp. In some embodiments,the plurality of
`oligonucleotides on the substrate deviates from a predetermined sequencespecific for each
`location with an error rate of less than 1/2000 bp.
`
`[0012]
`241 In practicing any of the methods of synthesizing n-mer oligonucleotides on a substrate as
`provided herein, in some embodiments, the building block comprises an adenine, guanine,
`thymine, cytosine, or uridine group. , Or a modified nucleotide is included. In some
`embodiments,the building block comprises modified nucleotides. In some embodiments, the
`building block comprises dinucleotides or trinucleotides. In some embodiments, the building
`block comprises a phosphoramidite. In some embodiments, n of the n-meroligonucleotideis
`at least 100. In some embodiments, n is at least 200. In some embodiments, n is at least 300.
`
`21-07-2020
`
`6
`
`

`

`In some embodiments, n is at least 400. In some embodiments, the surface comprises at least
`100,000 degraded sites and at least two of the plurality of growing oligonucleotides can
`differ from each other.
`
`[0013]
`254 In some embodiments, the method of synthesizing n-mer oligonucleotides on a substrate as
`described herein further comprises vacuum drying the substrate prior to conjugation. In
`some embodiments, the building block comprises a blocking group. In some embodiments,
`the blocking group comprises acid labile DMT. In some embodiments,the acid labile DMT
`comprises 4,4’-dimethoxytrityl. In some embodiments, the method of synthesizing an n-mer
`oligonucleotide on a substrate as described herein further comprises oxidation or sulfidation.
`In some embodiments, the method of synthesizing n-mer oligonucleotides on a substrate as
`described herein further comprises the step of chemically capping the separated
`oligonucleotide strands. In some embodiments, a method of synthesizing an n-mer
`oligonucleotide on a substrate as described herein removes the blocking group, thereby
`deblocking the growing oligonucleotide chain. The method further includes a deblocking
`step. In some embodiments, the position of the substrate during the bonding processis
`within 10 cm of the position of the substrate during the vacuum drying process. In some
`embodiments, the position of the substrate during the bonding step is within 10 cm of the
`position of the substrate during the oxidizing step. In some embodiments,the position of the
`substrate during the bonding processis within 10 cm of the position of the substrate during
`the cap process. In some embodiments, the position of the substrate during the bonding step
`is within 10 cm of the position of the substrate during the deblocking step. In some
`embodiments, the substrate comprises at least 10,000 vias that provide fluid communication
`between the first surface of the substrate and the second surface of the substrate. In some
`
`embodiments, the substrate comprises at least 100,000 vias that provide fluid
`communication between the first surface of the substrate and the second surface of the
`
`substrate. In some embodiments, the substrate includes at least 1,000,000vias that provide
`fluid communication between the first surface of the substrate and the second surface of the
`
`substrate.It is noted that any of the embodiments described herein may be combined with
`any of the methods, devices or systems provided herein.
`
`[0014]
`283 In another aspect of the invention, provided herein is a system for conducting a series of
`concurrent reactions. The system includesa first surface having a plurality of decomposed
`locations and a cap element having a plurality of decomposed reactor caps. In some
`embodiments, the system includesa plurality of disassembled reactor caps anda plurality of
`disassembled locations on thefirst surface forming a temporary seal betweenthefirst
`surface and the cap element. Aligning, thereby physically dividing the locations on the first
`surface into groupsof at least two locations into reactors associated with each reactor cap.In
`
`21-07-2020
`
`7
`
`

`

`some embodiments, each reactor holdsa first set of reagents.
`
`[0015]
`294 In some embodimentsrelating to any of the systems for performing a series of concurrent
`reactions as described herein, after release from the first surface, the reactor cap is at least
`one ofthefirst set of reagents. Hold a part. In some embodiments, the portion is about 30%.
`In some embodiments, the portion is about 90%. In some embodiments,the plurality of
`degradedsites are on microstructures created on the surface of the support. In some
`embodiments,the plurality of resolved locations has a density of at least 1 per mm<2>. In
`some embodiments,the plurality of resolved locations has a density of at least 10 per
`mm<2>. In some embodiments,the plurality of resolved locations is a density of at least 100
`per mm<2>. In some embodiments, the microstructure comprises at least two channels in
`fluid communication with each other. In some embodiments,the at least two channels
`include two channels having different widths. In some embodiments, the at least two
`channels include two channels having different lengths. In some embodiments,at least one of
`the channels is longer than 100 sm. In some embodiments,at least one of the channels is
`shorter than 1000 um. In some embodiments,at least one of the channels has a diameter
`greater than 50 um. In some embodiments,at least one of the channels is less than 100 “#m
`in diameter. In some embodiments, the system further comprises a second surface having a
`plurality of resolved sites at a density of at least 0.1 per mm<2>. In some embodiments,the
`system further comprises a second surface having a plurality of resolved sites at a density of
`at least 1 per mm<2>. In some embodiments, the system further comprises a second surface
`having a plurality of resolved sites at a density of at least 10 per mm<2>.
`
`[0016]
`317 In some embodimentsrelating to any of the systems for conducting a series of concurrent
`reactions as described herein, the degraded location of the first surface comprises a coating
`of reagents. In some embodiments, the degraded location of the second surface comprises a
`coating of reagents. In some embodiments, the coating of reagents is covalently attached to
`the first or second surface. In some embodiments, the reagent coating comprises an
`oligonucleotide. In some embodiments, the reagent coating has a surface area of at least 1.45
`im <2> per 1.0 4m <2> of planar surface area. In some embodiments, the reagent coating
`has a surface area of at least 1.25 m<2> per 1.0 4m<2> of planar surface area. In some
`embodiments, the reagent coating has a surface area of at least 1 #m <2> per 1.0 “wm <2>
`of planar surface area. In some embodiments, the decomposedsites in the plurality of
`decomposedsites include a nominal arc length of the perimeter with a density of at least
`0.001 m/ tm <2>. In some embodiments, the decomposedsites in the plurality of
`decomposedsites include a nominal arc length of the perimeter with a density of at least
`0.01 “m/ fm <2>. In some embodiments, the decomposedsites of the plurality of
`decomposedsites of the first surface include high energy surfaces. In some embodiments, the
`
`21-07-2020
`
`8
`
`

`

`first and second surfacesinclude different surface tensions than the applied liquid. In some
`embodiments, high surface energy correspondsto a water contact angle of less than 20
`degrees. In some embodiments,the plurality of degraded sites are located on a solid
`substrate comprising a material selected from the group consisting of silicon, polystyrene,
`agarose, dextran, cellulosic polymers, polyacrylamide, PDMS, and glass. In some
`embodiments, the cap element comprises a material selected from the group consisting of
`silicone, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamide, PDMS, and glass.
`It is noted that any of the embodiments described herein may be combined with any of the
`methods, devices or systems provided herein.
`
`[0017]
`344 In yet anotheraspect, the invention also provides an array of housings. The series of
`enclosures includes a plurality of disassembled reactors, including a first substrate and a
`second substrate that include reactor caps, and at least two disassembled locations in each
`reactor. In some cases, the disassembled reactors are separated by a peelable seal. In some
`cases, the reactor cap retains at least a portion of the reactor contents after ejection of the
`second substrate from thefirst substrate. In some embodiments, the reactor cap on the
`second substrate has a density of at least 0.1 per mm<2>. In some embodiments, the reactor
`cap on the second substrate has a density of at least 1 per mm<2>. In some embodiments,
`the reactor cap on the second substrate has a density of at least 10 per mm<2>.
`
`[0018]
`356 In some embodiments relating to a series of enclosures as provided herein, the reactor cap
`retains at least 30% of the reactor contents. In some embodiments, the reactor cap retains at
`least 90% of the reactor contents. In some embodiments, the resolved location is a density of
`at least 2/mm<2>. In some embodiments, the resolved location is a density of at least
`100/mm<2>. In some embodiments, the series of enclosures further comprisesat least 5
`disassembled locations in each reactor. In some embodiments, the series of enclosures as
`described herein further comprises at least 20 disassembled locations in each reactor. In
`some embodiments, the series of enclosures as described herein further comprises at least
`50 disassembled locations in each reactor. In some embodiments, the series of enclosures as
`described herein further comprises at least 100 disassembled locations in each reactor.
`
`[0019]
`369 In some embodimentsrelating to a series of housings as described herein, the disassembled
`location is on a microstructure created on the surface of the support. In some embodiments,
`the microstructure comprises at least two channels in fluid communication with each other.
`In some embodiments,the at least two channels include two channels having different
`widths. In some embodiments, the at least two channels include two channels having
`
`21-07-2020
`
`9
`
`

`

`different lengths. In some embodiments,at least one of the channels is longer than 100 “m.
`In some embodiments, at least one of the channels is shorter than 1000 ym. In some
`embodiments,at least one of the channels has a diameter greater than 50 “£m. In some
`embodiments,at least one of the channels is less than 100 “ m in diameter. In some
`embodiments, the microstructure comprises a nominal arc length around the perimeterof at
`least two channels having a density of at least 0.01 4 m/square ym. In some embodiments,
`the microstructure comprises a nominal arc length around the perimeter of at least two
`channels having a density of at least 0.001 4m/square 4m. In some embodiments, the
`disassembled reactors are separated by a peelable seal. In some embodiments,the peelable
`seal comprises a capillary burst valve.
`
`[0020]
`387 In some embodimentsrelating to a series of housings as described herein, the plurality of
`disassembled locations on the first substrate include a coating of reagents. In some
`embodiments,the plurality of resolved locations on the second substrate include a coating of
`reagents. In some embodiments, the coating of reagents is covalently attachedto thefirst or
`second surface. In some embodiments, the reagent coating comprises an oligonucleotide.In
`some embodiments, the reagent coating has a surface areaof at least 1 “im <2> per 1.0 wm
`<2> of planar surface area. In some embodiments, the reagent coating has a surface area of
`at least 1.25 m<2> per 1.0 & m<2> of planar surface area. In some embodiments, the
`reagent coating has a surface area of at least 1.45 “4m <2> per 1.0 “m <2> of planar
`surface area. In some embodiments, the plurality of resolved locations on the first substrate
`include high energy surfaces. In some embodiments,the first and second substrates include
`different surface tensions than the applied liquid. In some embodiments, the surface energy
`correspondsto a water contact angle of less than 20 degrees. In some embodiments, the
`plurality of degraded locations or reactor caps are on a solid substrate comprising a material
`selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic
`polymers, polyacrylamide, PDMS, and glass. Positioned. It is noted that any of the
`embodiments described herein may be combined