the mirna quality control study mirqc Jo Vandesompele Pieter Mestdagh

Transcription

1 the mirna quality control study mirqc Jo Vandesompele Pieter Mestdagh

2 The mirqc Consortium dr. Toumy Guettouche, University of Miami Novartis AIM Objective assessment of mirna profiling platforms qpcr hybridiza?on sequencing Life Technologies Agilent Illumina Exiqon Affymetrix Life Technologies Qiagen Quanta Biosciences Exiqon Nanostring Wafergen

3 mirqc study design set of 23 samples, profiled by each participant data analysis by mirqc consortium

4 mirqc study design sample set 1 4 MAQC samples, replicate measurements MAQC A : 100 % Universal Reference RNA MAQC B : 100 % Human Brain RNA MAQC C : 75 % MAQC A + 25 % MAQC B MAQC D : 25 % MAQC A + 75 % MAQC B titration response reproducibility 120" 100" 80" 60" 40" 20" 0" A" C" D" B" C/A = B/A D/A = B/A expression" 6" 5" 4" 3" 2" 1" 0" 0" 1" 2" 3" 4" 5" 6" expression"

5 mirqc study design sample set 2 MAQC B + gdna, replicate measurements mirqc study design sample set 3 synthetic mirnas spiked in RNA samples let-7 family hsa-let-7a: UGAGGUAGUAGGUUGUAUAGUU hsa-let-7b: UGAGGUAGUAGGUUGUGUGGUU hsa-let-7c: UGAGGUAGUAGGUUGUAUGGUU hsa-let-7d: AGAGGUAGUAGGUUGCAUAGUU mir-302 family hsa-mir-302a: UAAGUGCUUCCAUGUUUUGGUGA hsa-mir-302b: UAAGUGCUUCCAUGUUUUAGUAG hsa-mir-302c: UAAGUGCUUCCAUGUUUCAGUGG hsa-mir-302d: UAAGUGCUUCCAUGUUUGAGUGU

6 mirqc study design sample set 4 MS2 phage RNA, replicate measurements mirqc study design sample set 5 RNA from human serum, replicate measurements spiked with synthetic mirnas at low but varying concentrations sample 1 sample 2 mirna concentra?on mirna concentra?on mir- 10a 1 mir- 10a 5 let- 7a 1 let- 7a 2 mir- 302a 1 mir- 302a 0.5 mir- 133a 1 mir- 133a 0.2

7 sample set 1 - reproducibility A expression ( log 2 ) expression ( log 2 ) expression ( log 2 ) cumulative fraction (%) cumulative fraction (%) C 6 A replicate 14 expression expression difference ( log ( log 2 ) ) expression ( expression log 2 ) ( log 2 ) cumulative fraction cumulative (%) fraction (%) B expression ( log 2 ) replicate expression expression difference ( log( 2 log ) 2 ) B frequency frequency B frequency frequency B expression ( log 2 ) 0 expression ( log 2 )

8 sample set 1 Titra?on Response (4) (18) (47) (76) (18) (5) (10) (9) 0.8 titrating mirnas (%) titrating mirnas (%) (8) (14) (52) (78) (26) (18) (19) (37) binned difference ( log 2 ) MAQC A MAQC B binned difference ( log 2 ) MAQC A MAQC B

9 sample set 1 Titra?on Response MAD expect = median( y measured -y expect )/0.675 MAD fit = median( y measured -y fit )/ D/A = B/A MADexpect = MADfit = D/A 10 D/A B/A B/A

10 sample set 1 Titra?on Response D/A = B/A MADexpect = MADfit = D/A 10 D/A B/A B/A

11 sample set 3 - Specificity Table 1: mir-302 spike-in experiment mir-302a-3p mir-302b-3p mir-302c-3p mir-302d-3p mir-302a-3p mir-302b-3p mir-302c-3p mir-302d-3p Table 1: mir-302 spike-in experiment mir-302a-3p mir-302b-3p mir-302c-3p mir-302d-3p mir-302a-3p mir-302b-3p mir-302c-3p mir-302d-3p

12 sample set 3 - Specificity Table 2: let-7 spike-in experiment let-7a-5p let-7b-5p let-7c let-7d-5p let-7a-5p let-7b-5p let-7c let-7d-5p let-7e-5p let-7f-5p let-7g-5p let-7i-5p Table 2: let-7 spike-in experiment let-7a-5p let-7b-5p let-7c let-7d-5p let-7a-5p let-7b-5p let-7c let-7d-5p let-7e-5p let-7f-5p let-7g-5p let-7i-5p

13 sample set 5 Sensi?vity Serum samples low mirna content mirna detection rate in serum ~ measure sensitivity #"detected"mirnas" 120" 100" 80" 60" 40" 20" 0" 1" 2" 3" 4" 5" 6" 7" 8" 9"

14 low- copy template detec?on in serum relative expression (log2) relative expression (log2) mir 10a 5p let 7a 5p mir 302a 3p mir 133a mir 10a 5p let 7a 5p mir 302a 3p mir 133a

15 General Conclusions qpcr: sensitivity hybridization: reproducibility sequencing: content (isomirs) Manuscript in preparation (submitted early 2013)

16 courtesy of Gary Schroth, Illumina Expression level mature micro, mir*, and iso-forms Five different human tissue samples Hairpin sequence and loop structure hsa-mir-26b (-40.10) CCGGGACCCAGTTCAAGTAATTCAGGATAGGTTGTGTGCTGTCCAGCCTGTTCTCCATTACTTGGCTCGGGGACCGG ((((..((((((.((((((((..(((((((((((.(..()..))))))))))))..))))))))))).)))..)))) TTCAAGTAATTCAGGATAGGT TTCAAGTAATTCAGGATAGGTT TTCAAGTAATTCAGGATAG TTCAAGTAATTCAGGAT TTCAAGTAATTCAGGATA TTCAAGTAATTCAGGA TTCAAGTAATTCAGGATAGGTTG TTCAAGTAATTCAGGATAGGTTGT TTCAAGTAATTCAGGATAGG TCAAGTAATTCAGGATAGG TCAAGTAATTCAGGATAGGT TCAAGTAATTCAGGATAGGTT CAAGTAATTCAGGATAGG CAAGTAATTCAGGATAGGTT AAGTAATTCAGGATAGGTT AAGTAATTCAGGATAGG AGTAATTCAGGATAGGTT TAATTCAGGATAGGTT GTGTGCTGTCCAGCCTGTTC CCTGTTCTCCATTACTTGGC CCTGTTCTCCATTACTTGG CCTGTTCTCCATTACTTGGCTC CCTGTTCTCCATTACTTGGCT Mature mir*

17 courtesy of Gary Schroth, Illumina Isoform with higher expression level than mature micro mature forms based on mirbase V17 hsa-mir-30a (-37.30) GCGACTGTAAACATCCTCGACTGGAAGCTGTGAAGCCACAGATGGGCTTTCAGTCGGATGTTTGCAGCTGC (((.((((((((((((..((((((((((((((.().)))...))))))))))))))))))))))).))) -----TGTAAACATCCTCGACTGGAAG TGTAAACATCCTCGACTGGA TGTAAACATCCTCGACTGGAAGC TGTAAACATCCTCGACTGGAAGCT TGTAAACATCCTCGACTGGAA REST_OF_THE_ISOFORMS Total Counts 253, ,235 1,079, , ,750

18 RNA sequencing Option 1: Poly-A, non-directional Option 2: Ribo-depletion, directional

19 courtesy of Gary Schroth, Illumina

20 courtesy of Gary Schroth, Illumina