(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(10) International Publication Number
`
`WO 2018/057820 A1
`
`h a
`
`WIPOI PCT
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`29 March 2018 (29.03.2018)
`
`OM, PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report (Art. 21 (3))
`
`(51)
`
`International Patent Classification:
`
`CIZN 15/10 (2006.01)
`CIZQ 1/68 (2006.01)
`
`C40B 40/06 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2017/052832
`
`(84)
`
`(22)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`International Filing Date:
`21 September 2017 (21.09.2017)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`21 September 2016 (21.09.2016) US
`62/397,923
`PCT/US2017/027830
`16 April 2017 (16.04.2017)
`
`US
`
`[US/US]; 3555 Ardcn
`Applicant: PREDICINE, INC.
`Road, Hayward, California 94545 (US).
`
`Inventors: WANG, Xiaohong; 6620 S Mariposa Ln,
`Dublin, California 94568 (US). DU, Pan; 3211 Oak Bluff
`Ln, Dublin, California 94568 (US). JIA, Shidong; 399 Mar-
`garita Avenue, Palo Alto, California 94306 (US).
`
`Agent: SUN-HOFFMAN, Lin; PO. Box 52048, Palo Alto,
`California, 94303 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`A0, AT, AU, AZ, BA, BB, BG, BH, BN, BR, Bw, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
`HR, HU, 1D,1L, 1N, 1R, 1S, JO, JP, KE, KG, KH, KN, KP,
`KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`
`(54) Title: SYSTEMS AND METHODS FOR COMBINED DETECTION OF GENETIC ALTERATIONS
`
`FIG. 1
`
`RT and library
`,,
`
`,
`
` Nuddc; dd
`
`335: nequenmdg
`preparazlon
`extraction Rafi
`L::::::) “,E‘QA 122:1} Egg“; 5:)
`& Dunn
`”‘" Wich—layar
`w
`barcodes
`
`~
`”‘1: ,
`
`.
`
` Bidinfdrmaisds
`
`anaiysus
`
`/ DNAudants
`
`(57) Abstract: Disclosed are systems and methods for simultaneous detection of DNA and RNA genetic alterations comprising gene
`splicing variants, mutations, indel, copy number changes, fusion and combination thereof, in a, biofluid sample from the patient with-
`out physically separating RNA from DNA. The systems and methods are similarly applicable to the simultaneous detection of DNA
`and RNA genetic alterations in solid tissues comprising gene splicing variants, mutations, indel, copy number changes, fusion and
`combination thereof. The present method utilized a, barcoding method for analysis. The streamlined methods improve the simplicity,
`quantification accuracy and detection sensitivity and specificity of non-invasive detections of biomarkers.
`
`
`
`wo2018/057820A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`

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`WO 2018/057820
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`PCT/US2017/052832
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`SYSTEMS AND METHODS FOR COMBINED DETECTION OF GENETIC
`
`ALTERATIONS
`
`FIELD OF INVENTION
`
`[1]
`
`The invention relates generally to the field of precision medicine, specifically cancer
`
`prediction, diagnostics or prognostics, and, more specifically of Gene RADAR (RNA and DNA
`
`digital Reading) and NGS methods for detecting cancer mutations in a cancer patient by the
`
`simultaneous detection of genetic alterations including RNA splicing variants, DNA— and/or
`
`RNA—based mutation, indel, long deletions, copy number variation, gene fusions from solid
`
`tissues or biofluid samples, e. g., plasma, serum, urine, and saliva etc.
`
`BACKGROUND
`
`[2]
`
`Cancer is one of the leading causes of morbidity and mortality worldwide, with
`
`approximately 14 million new cases in 2012. The number of new cases is expected to rise by
`
`about 70% over the next 2 decades. The standard treatments of cancer include chemotherapy,
`
`irradiation therapy, surgery and more recently immune therapy. Detecting genetic alterations is a
`
`key step for cancer diagnosis and personalized medicine, thus genetic alteration screening has the
`
`potential to improve the overall healthcare of cancer patients.
`
`[3]
`
`Liquid biopsy including blood analysis is especially useful for cancer diagnosis because
`
`of the accessibility issue of the tumor and to avoid repetitive tumor biopsy during the course of
`
`treatments. Both DNA and RNA carries important genetic variant information for cancer
`
`diagnosis. For example, DNA can detect DNA mutations, DNA copy number and structure
`
`variations, while RNA can detect gene splicing and fusions, and RNA expression changes and
`
`also confirm DNA level mutations if they are expressed at RNA level. Therefore, there is a great
`
`need for detection of both DNA and RNA alterations from the same sample. The conventional
`
`way to detect both DNA and RNA alterations has to first physically separate RNA from DNA.
`
`However, the separation of RNA from DNA can cause material loss for both DNA and RNA,
`
`and the separation process is also cost and time consuming. Here we invent a solution of detect
`
`both DNA and RNA alterations without needing to physically separate RNA from DNA.
`
`

`

`WO 2018/057820
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`PCT/USZOl7/052832
`
`[4]
`
`Next generation sequencing (NGS) is accelerating the discovery of genetic alteration in
`
`human diseases. One of the major advantage of next generation sequencing is multiplex
`
`sequencing. NGS technology also enables adding molecular barcode to identify the source of the
`
`NGS reads.
`
`SUMMARY OF THE INVENTION
`
`[5]
`
`The present invention provides a method for detecting a genetic alteration from a biofluid
`
`comprises: a) obtaining nucleic acids comprising a single strand RNA (ssRNA) and a double
`
`strand DNA (dsDNA) from said biofluid; b) labeling ssRNA with a first barcode during reverse
`
`transcription step and converting ssRNA to double stranded cDNA wherein the ds—CDNA
`
`comprises RNA—specific barcodes, while the unbarcoded dsDNA is in the same tube; and c)
`
`labeling the DNA mixture comprising the unbarcoded dsDNA and the barcoded ds—cDNA with a
`
`second barcode; and d) analyzing the genetic alteration by a bioinformatics tool by deciphering
`
`two-layer RNA molecular barcoding. The present invention comprises a step of sequencing
`
`barcoded RNA and DNA simultaneously. The present invention further comprises a step of
`
`analyzing RNA and DNA sequencing results for detecting a genetic alteration after the
`
`sequencing.
`
`[6]
`
`The bioinformatics analysis enables l) differentiation of the RNA derived reads from
`
`DNA derived reads by checking the RNA specific tags in the sequence reads; 2) the suppression
`
`of the sequencing and background noise by creating consensus of Next Generation Sequence
`
`(NGS) reads from the same original molecules, which is defined based on molecular barcodes
`
`and the mapping location of the reads; and 3) accurate quantification of RNA by combining two
`
`types of barcodes (RNA molecular barcodes + DNA molecular barcodes), and the quantification
`
`of DNA (only using DNA molecular barcodes) at the same time.
`
`[7]
`
`The present invention further provides that reverse transcription step of the barcoded
`
`ssRNA comprises 1) reverse transcribing ssRN A to cDNA after ssRN A is annealed to an
`
`oligonucleotide comprising a RNA specific tag and random molecular barcodes; and 2)
`
`converting the cDNA to a ds—cDNA, wherein such barcoding step is named single—sided RNA
`
`barcoding.
`
`[8]
`
`The present invention further provides that reverse transcription step of the barcoded
`
`ssRNA, wherein the converting step of the cDNA to a ds—cDNA further comprising a second
`
`2
`
`

`

`WO 2018/057820
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`PCT/USZOl7/052832
`
`oligonucleotide with molecular barcode, wherein such barcoding step is named double—sided
`
`RNA barcoding.
`
`[9]
`
`In some embodiments, the barcoded DNA mixture is subsequently analyzed by Next
`
`Generation Sequencing.
`
`[10]
`
`In some embodiments, the genetic alterations to be detected comprising one or more gene
`
`splice variants, mutations, indels, long deletions, copy numbers changes, fusions and
`
`combination thereof.
`
`[11]
`
`The biofluid samples are selected from a group consisting of blood, plasma, serum, urine,
`
`sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the
`
`respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the
`
`lymphatic system, semen, cerebrospinal fluid, intra—organ system fluid, ascetic fluid, tumor cyst
`
`fluid, amniotic fluid, and a combination thereof. In preferred embodiments, the nucleic acid
`
`sample can be obtained by extracting both DNA and RNA from the biofluid sample
`
`simultaneously.
`
`[12]
`
`The present invention further comprises the detection of presence and absence of a
`
`genetic alteration is indicative of a disease and the disease is one or more cancers, for example, a
`
`genetic alteration is from an androgen receptor gene mutation which predicts prostate cancer. Or
`
`a genetic alteration is from lung panel gene mutation which predicts lung cancer.
`
`[13]
`
`The present invention further provides a platform/system for detecting genetic alterations
`
`in a patient, comprising: (a) a kit of reagents for circulating nucleic acid extraction and
`
`oligonucleotides targeting one or more gene alterations without separating RNA and DNA in
`
`said nucleic acid extraction; and (b) bioinformatics analysis solution to decipher DNA and RNA—
`
`derived information.
`
`[14]
`
`The system can be a closed system and an automated system.
`
`[15]
`
`Disclosed are systems and methods for detecting genetic alterations in lung, breast,
`
`ovarian, prostate and other cancer patients. In one aspect, the disclosed method comprises
`
`assaying the presence or absence of one or more gene splice variants and additional genetic
`
`alterations such as mutation, indels, long deletions, copy number variation, gene fusions etc. in a
`
`biofluid sample from the said patient.
`
`[16]
`
`The present invention provides a two—layer RNA molecular barcoding method for labeling
`
`a nucleic acid sample including a step for RNA molecular barcoding comprising adding a first
`
`

`

`WO 2018/057820
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`PCT/USZOl7/052832
`
`RNA specific molecular barcodes, wherein said RNA specific molecular barcodes are incorporated
`
`into a ds—CDNA converted from a ssRNA; and a step for DNA molecular barcoding comprising
`
`adding a second molecular barcodes by ligation of oligo adaptor to barcoded and unbarcoded
`
`dsDNA in said nucleic acid sample for further genetic alteration analysis.
`
`[17]
`
`The present invention further provides a system for detecting a genetic alteration of from
`
`a biofluid comprises: a) reagents for circulating nucleic acid extraction; b) barcoding sequences
`
`for two—layer RNA molecular barcoding; and c) bioinformatics tool to analyzing DNA and RNA—
`
`derived information.
`
`[18]
`
`In some embodiments, the biofluid sample is a sample of blood, plasma, serum, urine,
`
`sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the
`
`respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the
`
`lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascetic fluid, tumor cyst
`
`fluid, amniotic fluid, or a combination thereof.
`
`[19]
`
`In some embodiments, the step of assaying comprises extracting RNA from the biofluid
`
`sample and subsequently reverse transcribing the extracted RNA into a complementary DNA.
`
`[20]
`
`In other embodiments, the step of assaying comprised extracting both DNA and RNA
`
`from the biofluid sample simultaneously and then reverse transcribing the extracted RNA to a
`
`complementary DNA.
`
`[21]
`
`In some embodiments, the resultant complementary DNA is subsequently measured by
`
`Next Generation Sequencing, Polymerase Chain Reaction including qPCR and digital PCR,
`
`array-based technologies, and other related technologies.
`
`[22]
`
`The present invention further disclosed that Gene RADAR (RNA and DNA single
`
`molecular digital Reading) bioinformatics analysis tool can decipher two—layer RNA molecular
`
`barcoding to: (1) enable differentiation of the RNA derived reads from DNA derived reads by
`
`checking the RNA specific tags in the sequence reads; (2) enable the suppression of the sequencing
`
`and PCR errors by creating consensus of NGS reads from the same original molecular (based on
`
`molecular barcodes and the mapping location of the reads); (3) enable the quantification of RNA
`
`by combining two types of barcodes (RNA molecular barcodes + DNA molecular barcodes), and
`
`the quantification of chNA (only using DNA molecular barcodes) at the same time. Because the
`
`highly expressed genes need much higher barcode diversity to identify all unique RNA fragments
`
`

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`WO 2018/057820
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`PCT/US2017/052832
`
`than chNA, the two—layer barcode scheme combined with Gene RADAR analysis tool enables
`
`simultaneous detection of RNA and DNA genetic alterations from a single sample.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[23]
`
`Figure 1 shows a Gene RADAR (RNA and DNA single molecular digital Reading) assay
`
`flowchart depicting the steps from biofluid or tissue to wet lab testing and data analysis of RNA—
`
`and DNA—derived genetic alterations.
`
`[24]
`
`Figure 2 shows variant allele frequency analysis before and after barcode consensus noise
`
`suppression.
`
`[25]
`
`Figure 3 shows ctDNA detection sensitivity and accuracy analysis.
`
`[26]
`
`Figure 4 shows “RNA+DNA” combined detection has better coverage than chNA—
`
`based detection alone.
`
`[27]
`
`Figure 5 shows a mutation detected at both DNA and RNA level in a clinic plasma
`
`sample.
`
`[28]
`
`Figure 6 shows mutation landscape from the clinical samples. All marked mutation
`
`represent change in the protein sequences and function.
`
`[29]
`
`Figures 7A—7C show embodiments of two—layer RNA+DNA molecular barcoding
`
`scheme. (A)RT with single—side barcodes; (B) RT with double—side barcodes; (C) Ligate
`
`barcoded adapters to double stranded DNA
`
`[30]
`
`Figure 8 shows the Gene RADAR Bioinformatics analysis workflow.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[31]
`
`The present invention provides a method for combined detection of genetic alterations
`
`from a biofluid. The present invention also provides a two—layer RNA molecular barcoding
`
`method for labeling RNA and DNA in nucleic acids mixture from the biofluid samples for
`
`further genetic analysis.
`
`[32]
`
`DEFINITIONS
`
`[33]
`
`The term "about," particularly in reference to a given quantity, is meant to encompass
`
`deviations of plus or minus five percent.
`
`

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`WO 2018/057820
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`PCT/USZOl7/052832
`
`H H
`
`H
`
`[34]
`
`As used in this application, including the appended claims, the singular forms "a,
`
`an,
`
`and "the" include plural references, unless the content Clearly dictates otherwise, and are used
`
`interchangeably with "at least one" and "one or more.”
`H H
`
`H H‘
`
`H H‘
`
`H H
`
`[35]
`
`As used herein, the terms "comprises,
`
`comprising,
`
`includes,
`
`1ncluding,
`
`contains,"
`
`"containing," and any variations thereof, are intended to cover a non—exclusive inclusion, such
`
`that a process, method, product—by—process, or composition of matter that comprises, includes, or
`
`contains an element or list of elements does not include only those elements but can include
`
`other elements not expressly listed or inherent to such process, method, product—by—process, or
`
`composition of matter.
`
`[36]
`
`The term "patient,” as used herein preferably refers to a human, but also encompasses
`
`other mammals. It is noted that, as used herein, the terms "organism," "individual," "subject," or
`
`"patient" are used as synonyms and interchangeably.
`
`[37]
`
`The term “genetic alteration” comprise gene splice variants, SNV, Indel, CNV, fusion
`
`and combination thereof.
`
`[38]
`
`The term circulating tumor DNA (ctDNA) or circulating tumor RNA (ctRNA) is tumor—
`
`derived fragmented DNA or RNA in the bloodstream that is not associated with cells. ctDN A or
`
`ctRNA should not be confused With cell—free DNA (chNA) or cell—free RNA (chNA), a
`
`broader term which describes DNA or RNA that is freely circulating in the bloodstream, but is
`
`not necessarily of tumor origin.
`
`[39]
`
`The term “barcoding” or “barcode” means using one or more oligonucleotides as
`
`tags/markers to incorporate into a dsDNA. The barcodes will be sequenced together with the
`
`unknown sample DNA. After sequencing the reads are sorted by barcode and grouped together
`
`(de—multiplexing). Barcode includes molecular barcode and sample barcode.
`
`[40]
`
`A “molecular barcode” is a unique multiple—base pair sequence used to identify unique
`
`fragments and “dc—duplicate” the sequencing reads from a sample. This, along with the random
`
`start sites, helps identify and remove PCR duplicates. Molecular barcodes can be used to
`
`suppress sequencing and PCR errors, and reduce false positives subsequently. Whereas sample
`
`barcodes, also called indexed adaptors, are customarily used in most current NGS workflows and
`
`allow the mixing of samples prior to sequencing.
`
`[41]
`
`The term “RNA molecular barcoding” means incorporating barcodes during the process
`
`of reverse transcription of RNA and dsDNA library preparation. RNA molecular barcoding can
`
`

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`WO 2018/057820
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`PCT/USZOl7/052832
`
`incorporate a molecular barcode or multiple molecular barcodes. A RNA specific barcode can be
`
`a RNA specific tag, a molecular barcode, a sample barcode or a combination.
`
`[42]
`
`The term “DNA barcoding” means barcoding at dsDNA level with a multiple—base pair
`
`sequence that is part of the adapter for multiplex sequencing. In some embodiment, the adapter is
`
`designed in house. The incorporated DNA barcodes can be molecular barcodes alone or both
`
`molecular barcodes and sample barcodes.
`
`[43]
`
`The term “positive” strand also known as the “sense” strand or coding strand, is the
`
`segment within double—stranded DNA that runs from 5' to 3'. The term “negative” strand also
`
`known as the “anti—sense” stand of DNA is the segment within double—stranded DNA that runs
`
`from 3‘ to 5'.
`
`[44]
`
`The term “bioinformatics” means a sequencing analysis tool/software including but are
`
`not limited to Gene-RADAR software or any software that can analyze DNA/RNA sequencing
`
`results.
`
`[45]
`
`The present invention provides a method for barcoding an oligonucleotide tag on the
`
`RNA sample during reserve transcribing it to cDNA and ds—cDNA, wherein the reverse
`
`transcription step of the ssRN A includes 1) reverse transcribing ssRN A to cDNA using a gene—
`
`specific or random primer annealed to an oligonucleotide comprising a RNA specific tag and
`
`random molecular barcodes; and/or 2) converting the cDNA to a ds—cDNA. In some
`
`embodiments, converting the cDNA to a ds—cDNA is conducted by annealing a non—coded
`
`primer, wherein such barcoding step is named single-sided RNA barcoding. In one embodiment,
`
`the RNA specific tag comprising an oligo nucleotide. The random molecular barcodes comprise
`
`another oligonucleotide. The oligo nucleotide consists of 5, 8, 10, 12, 14, 15, 20 nucleic acid
`
`bases. In another embodiment, the oligo nucleotide can be designed for fitting the identification
`
`in further analysis.
`
`[46]
`
`In some other embodiments, the converting step of the cDNA to a ds—cDNA is conducted
`
`by annealing an oligonucleotide comprising a second RNA specific molecular barcode, wherein
`
`such barcoding step is named double—sided RNA barcoding. In some embodiment, the first and
`
`second RNA specific molecular barcode is the same; in some other embodiment, the first RNA
`
`and second RNA specific molecular barcode is not the same.
`
`[47]
`
`In preferred embodiments, the genetic alterations include gene splice variants, mutations,
`
`indels, long deletions, copy number changes, fusions and combination thereof. The method of
`
`

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`WO 2018/057820
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`PCT/USZOl7/052832
`
`detecting the alterations is used to detect the changes of above.
`
`[48]
`
`The present invention further disclosed a two—layer RNA molecular barcoding scheme for
`
`further genetic analysis to distinguish RNA and DNA derived signals. Two-layer RNA
`
`molecular barcoding can reduce the background noise and variants (Figure 2) after barcode
`
`consensus noise suppression. In preferred embodiments, the first layer RNA molecular barcoding
`
`scheme includes adding RNA specific tags plus random molecular barcodes, as shown in figure
`
`7A and figure 7B. Figure 7A shows the procedure of adding singlc—sidcd RNA molecular
`
`barcodes and converting the single stranded RNA fragment to double stranded cDN A. Figure 7B
`
`shows the procedure of adding double—sided RNA molecular barcodes. Comparing with single—
`
`sided molecular barcoding scheme, an additional 3’ end repair step is added to get the 3’
`
`molecular barcodes. After RNA is converted as ds—CDNA (double—stranded cDNA), it will be
`
`treated the same as regular ds-chNA. Then the DNA level barcodes are added by ligation of
`
`adaptors, as shown in Figure 7C.
`
`[49]
`
`In one embodiment, the barcoded signals from RNA and DNA are read by next
`
`generation sequencing, not polymerase chain reaction. Separation of RNA and DNA derived
`
`reads is conducted with a bioinformatics analysis tool Gene—RADAR without physically
`
`separating nucleic acid extraction into RNA and DNA samples. Therefore, there is no material
`
`loss of DNA or RNA due to the physical separation of RNA from DNA. This method also
`
`reduced the cost and processing time.
`
`[50]
`
`In another embodiment, after the next generation sequencing simultaneous reads signals
`
`from RNA and DNA, a database file of the RNA molecular barcodes and DNA molecular
`
`barcodes will be utilized to recognize the reads from RNA or DNA with barcode matches. Then
`
`the recognized DNA reads are mapped to genome, while the recognized RNA reads are mapped
`
`to transcriptome and genome. Barcode consensus are created by merging NGS reads originally
`
`from the same molecule (identified based on molecular barcodes and genome mapping location
`
`of the reads). The sequencing and PCR errors can be corrected or marked when there are
`
`inconsistent variants originally from the same molecule. In some embodiments, the genetic
`
`alteration of DNA includes SNV, Indel, long deletion, CNV and DNA fusion. In some
`
`embodiments, the genetic alteration of RNA includes splicing, fusion, SNV, Indel analysis. Then
`
`the DNA and RNA analysis results are integrated to achieve comprehensive reporting of genetic
`
`alterations .
`
`

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`WO 2018/057820
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`PCT/USZOl7/052832
`
`[51]
`
`In one embodiment, barcode or barcoding with random oligonucleotide sequences such
`
`as 5, 8, IO 12, l4, l5 nucleotides to uniquely tag individual target, DNA n’tolecules can be used.
`
`In another embodiment, the oligo nucleotide can be designed for fitting the identification in
`
`further analysis. Such application increases the sensitivity and reduce false positives. For
`
`example, it can be used for PCR or NGS analysis to identify individual molecules (DNA or RNA
`
`fragments) in samples.
`
`[52]
`
`In some embodiments, the Gene RADAR detects DNA copy number gains while
`
`measuring other RNA level variants including splicing, fusion, SNV, Indel at the same time from
`
`the patient biofluid sample.
`
`[53]
`
`Present invention further provides a platform for detecting multiple gcnc variants in a
`
`patient, including: (a) a kit of reagents for circulating nucleic acid extraction; (b) barcoding
`
`sequences for two—layer RNA+DNA molecular barcoding; and (c) bioinformatics tool to
`
`analyzing DNA and RNA—derived information.
`
`[54]
`
`The system can be an opened or closed system. And both systems can be automated
`
`system. The system can be in a device setting.
`
`[55]
`
`In preferred embodiments, the detection of presence and absence of a genetic alteration is
`
`indicative of a disease and the disease is one or more cancers. In some other embodiments,
`
`presence and absence of multiple genetic alteration is indicative of a disease and the disease is
`
`one or more cancers. In some embodiments, genetic alteration detection is lung cancer or
`
`prostate cancer oncogene variation.
`
`[56]
`
`In one embodiment, the samples include but are not limited to blood, plasma, serum,
`
`urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid
`
`of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from
`
`the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor
`
`cyst fluid, amniotic fluid, and a combination thereof.
`
`[57]
`
`In one aspect, the disclosed method also allows for the reverse transcribed RNA (cDNA)
`
`to be included in the mainstream chNA library preparation and target enrichment protocol
`
`thereby allowing consolidated result of both DNA and RNA from a single biofluid sample. For
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`example, chNA and chNA are extracted simultaneously, and then the extracted sample are
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`used for reverse transcription and further used for the library processing and sequencing. In the
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`process, there is no need to separate chNA from the chNA in the biofluid nucleic acid
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`extraction. In some instance, to distinguish the DNA— and RNA—derived signals, the extracted
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`RNA is barcoded with a one—sided or two—sided barcoding method and reverse transcribed in a
`
`single step before the steps of library preparation and sequencing.
`
`[58]
`
`In some embodiments, the detected genetic alteration information can be used to detect
`
`castration resistant prostate cancer in a patient comprising assaying the presence or absence of
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`one or more types of genetic alterations at both RNA and DNA levels, such as androgen receptor
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`gene RNA splice variants (AR—Vs) and RNA/DNA—bascd mutation detection in a biofluid
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`sample from the patient; wherein the presence of such genetic alterations indicates the presence
`
`of the castration resistant prostate cancer in the patient.
`
`[59]
`
`In some embodiments, RNA is extracted together with DNAs from circulating nucleic
`
`acid and nucleic acid—containing extracellular vesicles in a biofluid sample. No extra step of
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`RNA purification is needed and therefore the process is simplified.
`
`[60]
`
`In some embodiments, the sources of nucleic acids are extracellular vesicles (EVs),
`
`including exosomes and microvesicles, which have been shown to carry a variety of
`
`biomacromolecules including mRNA, microRNA and other non—coding RNAs and considered to
`
`be a minimally invasive novel source of materials for molecular diagnostics. See J ia et al.,
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`“Emerging technologies in extracellular vesicle—based molecular diagnostics”, Expert Rev. Mol.
`
`Diagn. 1—15 (2014). EVs are membranous, cell—derived, mixed populations of vesicles, ranging
`
`from approximately 40—5000 nm in diameter, which are released by a variety of cells into the
`
`intercellular microenvironment and various extracellular biofluids. Methods for procuring a
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`microvesicle fraction from a biofluid sample are described in scientific publications and patent
`
`applications (Chen et al., 2010; Miranda et al., 2010; Skog et al., 2008). See also WO
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`2009/100029, WO 2011009104, WO 2011031892, and WO 2011031877. For example, methods
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`of microvesicle procurement by differential centrifugation are described in a paper by Raposo et
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`al. (Raposo et al., 1996), a paper by Skog et al. (Skog et al., 2008) and a paper by Nilsson et. al.
`
`(Nils son et al., 2009). Methods of anion exchange and/or gel permeation chromatography are
`
`described in US. Pat. Nos. 6,899,863 and 6,812,023. Methods of sucrose density gradients or
`
`organelle electrophoresis are described in US. Pat. No. 7,198,923. A method of magnetic
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`activated cell sorting (MACS) is described in a paper by Taylor and Gercel-Taylor (Taylor and
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`Gercel—Taylor, 2008). A method of nanomembrane ultrafiltration concentration is described in a
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`paper by Cheruvanky et al. (Cheruvanky et al., 2007). Further, microvesicles can be identified
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`and isolated from a subject's bodily fluid by a microchip technology that uses a microfluidic
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`platform to separate tumor—derived microvesicles (Chen et al., 2010).
`
`[61] Methods for nucleic acid extraction are generally based on procedures well-known in the
`
`art plus proprietary procedures developed in-house. Persons of skill will select a particular
`
`extraction procedure as appropriate for the particular biological sample. Examples of extraction
`
`procedures are provided in patent publications WO/2009/ 100029, US 20100196426, US
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`20110003704, US 20110053157, WO 2011009104, WO 2011031892, US20130131194 and
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`US20110151460. Each of the foregoing references is incorporated by reference herein for its
`
`teaching of these methods.
`
`[62] Many biofluids contain circulating nucleic acids and/or nuclcic acid—containing EVs.
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`Examples of these biofluids include blood, plasma, serum, urine, sputum, spinal fluid,
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`cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal,
`
`and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen,
`
`cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, or a
`
`combination thereof.
`
`[63]
`
`In some embodiments, the biofluid sample is obtained from a subject who has been
`
`diagnosed with cancer based on tissue or liquid biopsy and/or surgery or clinical grounds.
`
`[64]
`
`It must be noted that, as used in this specification and the appended claims, the singular
`
`forms a , an" and "the" include plural referents unless the content clearly dictates otherwise.
`
`Thus, for example, reference to "a biomarker" includes a mixture of two or more biomarkers, and
`
`the like.
`
`Examples
`
`[65]
`
`Example 1: Variant Allele Frequency before and after barcode consensus noise
`
`suppression.
`
`[66]
`
`To compare the performance of Gene RADAR and traditional next—generation
`
`sequencing using Picard deduplication, which lacks a consensus calling feature, 1.25ng Horizon
`
`reference HD701 was spiked in to normal chN A to get 30ng mixed DNA as input for PrediSeq—
`
`Pan Cancer assay. Results from Gene RADAR’s analysis pipeline and traditional method were
`
`compared and are shown in Figure 2. Using the traditional method, there were 50,820 identified
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`variants (3,838 variants with variant frequency > 0.1%). After applying Gene RADAR’s
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`consensus error correction, only 1,104 variants were identified (642 variants with variant
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`frequency > 0.1%). This indicates that Gene RADAR’s consensus error correction reduces
`
`background noise by 97.8% (or 85.1% for variants with AF > 0.1%). It was demonstrated that
`
`Gene RADAR’ s error suppression feature enables ultra—high quality sequencing of each input
`
`molecule of chNA.
`
`[67]
`
`Example 2: ctDNA detection analysis sensitivity and accuracy.
`
`[68]
`
`To evaluate analytic sensitivity of the PrediSeq—Pan Cancer assay, spike—in of Horizon
`
`reference DNA (HD701) was used, and 6 different SNVs were chosen for analysis. Four serial
`
`dilution ratios at 8.3%, 4.2%, 2.1%, 1.0% were used in triplicate samples to make a total 72 SNV
`
`targets. The SNV allele frequency at which > 90% (18 of 20) of SNVs are detected is defined as
`
`the limit of detection, and it was calculated at 0.1% based on the data (Figure 3). To assess the
`
`analytical accuracy of the assay’s SNV AF detection, we analyzed this same set of data,
`
`choosing the SNVs with detected AF > 0.1% and calculated the correlation between detected and
`
`expected AF, at 0.938.
`
`[69]
`
`Example 3: “RNA-l-DNA” combined detection has better coverage than chNA
`
`detection alone and additional Sensitivity and Accuracy contributed by chNA utilization. Gene
`
`RADAR technology utilizes chNA and chNA from the same sample simultaneously, which
`
`adds additional sequencing coverage compared to chNA sequencing alone. For the proof of
`
`concept study, prostate cancer DNA and RNA was extracted and sequenced from Vcap cell line
`
`supernatant. The coverage differences of selected cancer related target genes in DNA only vs.
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`DNA+RNA are presented in Figure 3. The black dots represent the genes which have matched
`
`DNA and RNA level variants. Roughly 17.5% of genes in the selected panel benefits from at
`
`least 10% coverage, and thus higher sensitivity contributed by the chNA input. In additional,
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`46.9% of the mutations detected from chNA were also detected in the same location from
`
`chNA (supported by at least 3 reads with target mutation). Therefore, by combining DNA a