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(12) Patent Application Publication (10) Pub. No.: US 2015/0307874 A1 JAITIN Et Al US 20150307874A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0307874 A1 JAITIN et al. (43) Pub. Date: Oct. 29, 2015 (54) HIGH THROUGHPUT TRANSCRIPTOME Publication Classification ANALYSIS (51) Int. Cl. (71) Applicant: Yeda Research and Development Co. CI2N 5/10 (2006.01) Ltd, Rehovot (IL) CI2O I/68 (2006.01) (52) U.S. Cl. (72) Inventors: Diego JAITIN, Rehovot (IL); Ido Amit, CPC .......... CI2N 15/1065 (2013.01); C12O I/6806 Rehovot (IL); Hadas Keren-Shaul, (2013.01) Rehovot (IL); Liran Valadarsky, Rehovot (IL) (57) ABSTRACT Kits and methods for single cellor multiple cell transcriptome (21) Appl. No.: 14/795,039 analysis are provided. An adapter polynucleotide is disclosed which comprises a double-stranded DNA portion of 15 base (22) Filed: Jul. 9, 2015 pairs and no more than 100 base pairs with a 3" single stranded overhang of at least 3 bases and no more than 10 bases, Related U.S. Application Data wherein said double stranded DNA portion is at the 5' end of (63) Continuation-in-part of application No. PCT/IB2014/ the polynucleotide and wherein the sequence of said 3' single 058153 filed on Jan. 9, 2014. Stranded overhang is selected from the group consisting of s s SEQ ID NOs: 1-8 and 9, wherein the 5' end of the strand of (60) Provisional application No. 61/750,454, filed on Jan. said double-stranded DNA which is devoid of said 3' single 9, 2013. Stranded overhang comprises a free phosphate. Patent Application Publication Oct. 29, 2015 Sheet 1 of 30 US 2015/0307874 A1 Jeddin - JeMO cd Co. Cd Co o Cld co o Co o co co o go to r cy. CNE was - AISueue dueS Patent Application Publication Oct. 29, 2015 Sheet 2 of 30 US 2015/0307874 A1 009 000 001 009 96. 00 00 JeMO Cd O d C. Cd Co. Co O O. r CYo cN r in KISueue dueS Patent Application Publication Oct. 29, 2015 Sheet 3 of 30 US 2015/0307874 A1 Jedd 99. JeMO O O. O. C. Cd C do o cd r Crd on re n- KSueue dueS Patent Application Publication Oct. 29, 2015 Sheet 4 of 30 US 2015/0307874 A1 s S s 3. e 8838& Patent Application Publication Oct. 29, 2015 Sheet 5 of 30 US 2015/0307874 A1 00001 {{{}{}; 8£(943 £996^0=2×3 tes sssse's t Loooool ke:: Patent Application Publication Oct. 29, 2015 Sheet 6 of 30 US 2015/0307874 A1 input: total RNA Step 1: Fragmentation (-200nts) and polyAt selection Step 2: Reverse transcription + terminal deoxynucleotidyltransferase 5 A 3 CCC v. 3-arcie p. i8:8888 Step 3: Sample pooling Step 4: Rnase H treatment (RNAstrand and removal) 3 CCC 3:08:8-8: 5 8:888 Step 5: Adapter tigation Sense 5 00:38::38.8%: antisense or CCC : 8 5. Step 6: Amplification + lumina primer P1 addition (PCR) : -------------Legend: 5 x CCC x 3 i & RNA 3 O x 5' : 8 P2 antisense P_rd forward PCR primer : « Id 3' 83 5 & P2 SeSe a D H cDNA P2 reverse PCR primer : P. a a new SSDNA Library ready for Huimina sequencing FG. 4 Patent Application Publication Oct. 29, 2015 Sheet 7 of 30 US 2015/0307874 A1 F.G. 5 Single Cell TRANS-seq Day input. Single cell of extremely low amount of RNA Step 1: Reverse transcription 5 a A. 3 m; : -33::::::. Step 2: Sample pooling Step 3: Second strand synthesis or oligoannealing for VT priming Step 4: in Vitro Transcription (linear amplification} Tipolymerase barCOded RNA non- ---------- - cDNA w w w a a a as a s w w w r r - a a a a .8 Patent Application Publication Oct. 29, 2015 Sheet 8 of 30 US 2015/0307874 A1 CDC MO dis CDC B poC NK T uoneo||duleLAI Patent Application Publication Oct. 29, 2015 Sheet 9 of 30 US 2015/0307874 A1 09°5343 *::*·********** **** : ; e SE3 t ************** .* **x*x«...«* * &&888.8. Patent Application Publication Oct. 29, 2015 Sheet 10 of 30 US 2015/0307874 A1 09,0},0009000100z $33. easia eski, Patent Application Publication Oct. 29, 2015 Sheet 11 of 30 US 2015/0307874 A1 $838&idier8: V/A’943 Patent Application Publication Oct. 29, 2015 Sheet 12 of 30 US 2015/0307874 A1 CPO qeue deu.9 991)f WKOSOO 16XN 0X eu:ZS) A90 COO 0. Cyß.1893L190899 eyO80 25us till (ZK UOW £160 pSIO ddeS ddes uje3N eu) d;0 XOil Ji JLSO qb .0 obO ebo eOdy foe.8S degdy UONH S3 LeysN : e10 6 X600000. 1990 Siege (6100 Z80CA SUOyendodqS Patent Application Publication Oct. 29, 2015 Sheet 13 of 30 US 2015/0307874 A1 90XO “eas s Cul s O edS s 67.97W q9quideS e9 Lieut 0980S 8. UX N. 1dd pCid C eeeN E09 Zuieu. O ! U0S 08 C91.Uetii. infly du. e60CPO 90d 209/. 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Kix: 88: .338. assag 8:883:388x: E63EEEO CNG Patent Application Publication Oct. 29, 2015 Sheet 21 of 30 US 2015/0307874 A1 Patent Application Publication Oct. 29, 2015 Sheet 22 of 30 US 2015/0307874 A1 9-16 Patent Application Publication Oct. 29, 2015 Sheet 23 of 30 US 2015/0307874 A1 }{ { } } } } { { { } } { } } } } { { { &~~~~--~~~~--~~~~~--~~~~--~~~~~--~~~~ C16*943 Patent Application Publication Oct. 29, 2015 Sheet 24 of 30 US 2015/0307874 A1 -29CO-7CO +7GO +800 (HI)-8- Sesse|OOS () 8. UJESE 0 (III)-8- SºssepOS Patent Application Publication Oct. 29, 2015 Sheet 25 of 30 US 2015/0307874 A1 Step Reverse transcription 5 A 3 3. NTUM-barcode-partial rc2'-T7 promoter 5 Step 2: Exonuclease Step 3: Sample pooing Step 4: Second strand synthesis 3' NTUM-barcode-partial rc2'-T7 promoter 5' 5 8 8 x X x X x X x X x X x X x X x X x X x X x 8 x 8.8 x 8 8888 x - 3. Step 5: in Virto Transcription Step 6: DNase 3 8 insii-bar{3departiairc2.5 Step 7: RNA Fragmentation 8 : xiii. 8:88-8; d. 5’ Step 8: RNAssDNA ligation 8 iii-8-barcode-partia: t2 5' Step 9: Reverse transcription 3' 8 in-ii-bascade-patia c.25 5 888: 8388: 8888.888: 888: 88888 six 3. Step 40. Amplification + Eumina primers addition by nested PCR P5 fc. forward & · P7 rc2 reverse primer *x Library ready for illumina Sequencing FG. 1 O Patent Application Publication Oct. 29, 2015 Sheet 26 of 30 US 2015/0307874 A1 Z64-16 |------.-.-.-.«** 96-4 Patent Application Publication Oct. 29, 2015 Sheet 27 of 30 US 2015/0307874 A1 wasS2 8r as -o d i E r o CY c. as 9 a. - oldS. C.) o2s CO Patent Application Publication Oct. 29, 2015 Sheet 28 of 30 US 2015/0307874 A1 st-sessessessessessee s Patent Application Publication Oct. 29, 2015 Sheet 29 of 30 US 2015/0307874 A1 ######### 33;&çë Patent Application Publication Oct. 29, 2015 Sheet 30 of 30 US 2015/0307874 A1 px; : g : o : 'a & w ey e so tossedXaXN US 2015/0307874 A1 Oct. 29, 2015 HIGH THROUGHPUT TRANSCRIPTOME cost, by the need to amplify DNA through bacterial cloning, ANALYSIS and by the traditional Sanger approach of sequencing by chain termination. RELATED APPLICATIONS 10007. The next-generation sequencing (NGS) technology eliminates some of these barriers, enabling massive parallel 0001. This application is a Continuation in Part of PCT sequencing at a high but reasonable cost for small studies. The Patent Application No. PCT/IB2014/058153 having Interna technology essentially reduces the transcriptome to a series of tional filing date of Jan. 9, 2014, which claims the benefit of randomly fragmented segments of a few hundred nucleotides priority under 35 USC S119(e) of U.S. Provisional Patent in length. These molecules are amplified by a process that Application No. 61/750,454 filed on Jan. 9, 2013. The con retains spatial clustering of the PCR products, and individual tents of the above applications are all incorporated by refer clusters are sequenced in parallel by one of several technolo ence as if fully set forth herein in their entirety. gies. Current NGS platforms include the Roche 454 Genome Sequencer, Illumina’s Genome Analyzer, and Applied Bio SEQUENCE LISTING STATEMENT systems’ SOLiD. These platforms can analyze tens to hun dreds of millions of DNA fragments simultaneously, generate 0002. The ASCII file, entitled 63112SequenceListing..txt, giga-bases of sequence information from a single run, and created on Jul. 9, 2015, comprising 25,234 bytes, submitted have revolutionized SAGE and cDNA sequencing technol concurrently with the filing of this application is incorporated ogy. For example, the 3' tag Digital Gene Expression (DGE) herein by reference.
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