Synthetic microrna Reference Standards Genomics Research Group ABRF 2015

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1 Synthetic microrna Reference Standards Genomics Research Group ABRF 2015 Don A. Baldwin, Ph.D.

2 Reference samples for Platform evaluation Protocol development Assay service improvement

3 Randomize Key Name CLUSTALW 1 hsa-mir-664*...acuggcuagggaaaaugauuggau... 2 hsa-mir uagauaaaauauugguaccug... 3 hsa-mir-576-3p...aagauguggaaaa.auuggaauc... 4 hsa-mir-208a...auaagacgagcaaaaagcuugu... 5 hsa-mir-208b...auaagacgaacaaaagguuugu... 6 hsa-mir uaaag.uaaauaugcaccaaaa... 7 hsa-mir-9*...auaaagcuagaua...accgaaagu... 8 hsa-mir-142-5p...cauaaag.uagaaa.gcacuacu... 9 hsa-mir ucaagagcaauaacgaaaaaugu hsa-mir auggagauagauauagaaau hsa-mir-516a-5p...uucucgaggaaagaagcacuuuc hsa-mir-516b...aucuggagguaagaagcacuuu hsa-mir-1255a...aggaugagcaaagaaaguagauu hsa-mir-1255b...cggaugagcaaagaaagugguu hsa-mir uggaggag.aaggaaggugaug hsa-mir-483-5p...aagacgggaggaaagaagggag hsa-mir-1...uggaauguaaagaaguauguau hsa-mir uggaauguaaggaagugugugg hsa-mir-452*...cucaucugcaaagaaguaagug hsa-mir ucugggcaacaaagugagaccu hsa-mir gggcgacaaagcaagacucuuucuu hsa-mir gggacccagggagagacguaag hsa-mir-423-5p...ugaggggcagagagcgagacuuu hsa-mir ugcaggaccaagaugagcccu hsa-mir-96*...aaucaugugcagugccaauaug hsa-mir ggcuagcaacagcgcuuaccu hsa-mir acucuagcugccaaaggcgcu... Design of Standards Random selection across range of composition Four pools of 16 Fifth pool of 829 Distribution of selected family members Many thanks to Life Technologies - AB

4 Name Sequence Pool hsa-mir-130b...cagugcaaugaugaaagggcau... 1 hsa-mir-130a...cagugcaauguuaaaagggcau... 2 hsa-mir-301b...cagugcaaugauauugucaaagc... 3 hsa-mir-301a...cagugcaauaguauugucaaagc... 4 hsa-mir-376a...aucauagaggaaaauccacgu... 1 hsa-mir-376c...aacauagaggaaauuccacgu... 2 hsa-mir-181d...aacauucauuguugucggugggu... 3 hsa-mir-181b...aacauucauugcugucggugggu... 4 hsa-let-7f...ugagguaguagauuguauaguu... 1 hsa-let-7a...ugagguaguagguuguauaguu... 2 hsa-let-7e...ugagguaggagguuguauaguu... 3 hsa-let-7b...ugagguaguagguugugugguu... 4 hsa-let-7c...ugagguaguagguuguaugguu... 1 hsa-mir-98...ugagguaguaaguuguauuguu... 2 hsa-let-7d...agagguaguagguugcauaguu... 3 hsa-let-7g...ugagguaguaguuuguacaguu... 4 hsa-let-7i...ugagguaguaguuugugcuguu... 1 hsa-mir-520b...aaagugcuuccuuuuagaggg... 2 hsa-mir-520c-3p...aaagugcuuccuuuuagagggu... 3 hsa-mir-520f...aagugcuuccuuuuagaggguu... 4 hsa-mir-15b...uagcagcacaucaugguuuaca... 1 hsa-mir-15a...uagcagcacauaaugguuugug... 2 hsa-mir-23a...aucacauugccagggauuucc... 3 hsa-mir-23b...aucacauugccagggauuacc... 4 Target discrimination Related sequences assigned to separate pools, should be measured at different abundances within a Standard

5 Preparation of Standards Request protocol from Don Request shipment of pools Send a prepaid shipping label, or your address and FedEx account number Mix pools to create Standards A-E Standard Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 (majority) A B 0.5x C x D x 0. E x Total micrornas = 0.5 nm each mirna Each Standard contains 3 ng/ul

6 Affymetrix mirna GeneChip Sridar Chittur, SUNY Albany One chip per Standard RMA background correction No inter-chip normalization Calculate pool averages for each Standard Average signal Pool dilution

7 Affymetrix mirna GeneChip Discrimination of related mirnas Signal Standard Name Sequence Pool hsa-mir-130b...cagugcaaugaugaaagggcau... 1 hsa-mir-130a...cagugcaauguuaaaagggcau... 2 hsa-mir-301b...cagugcaaugauauugucaaagc... 3 hsa-mir-301a...cagugcaauaguauugucaaagc... 4

8 Affymetrix mirna GeneChip Cross-hybridization of related mirnas Signal Standard Name Sequence Pool hsa-mir-520b...aaagugcuuccuuuuagaggg... 2 hsa-mir-520c-3p...aaagugcuuccuuuuagagggu... 3 hsa-mir-520f...aagugcuuccuuuuagaggguu... 4

9 HTG Molecular Bruce Seligmann Debrah Thompson HTG EdgeSeq Standards spiked into yeast trna, 75 pg input Nuclease protection assay Quantitation by probe sequencing Bowtie2 alignment to mirbase mature mirnas in probe panel

10 ABRF reference mirna Latin Square Test 0.5x 0.5x 0.5x x mirbase 19 assay

11 Specificity differentiating between closely-related mirnas Pool-specific signals (using all probes) 1-base difference 2-base difference 6862 total counts total counts Pool 3 Pool 4 mir-301a-3p 30 mir-301b hsa-mir-301b CAGUGCAAUGAUAUUGUCAAAGC hsa-mir-301a CAGUGCAAUAGUAUUGUCAAAGC Pool 2 Pool 3 let-7e-5p let-7a-5p hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU EdgeSeq differentiates between closely-related mirnas (1 or 2 base differences) in different pools, even within a complex background.

12 Illumina Small RNA Sequencing Catharine Aquino Hubert Rehrauer Functional Genomics Center Zurich Standards only, no background TruSeq Small RNA Library Prep Kit HiSeq 2500 Alternative library production Adam Morris Bioo Scientific No 5 phosphates on the oligomers

13 Illumina Small RNA Sequencing

14 Illumina Small RNA Sequencing Read count Mapped length

15 Illumina Small RNA Sequencing Read count Mapped length

16 Illumina Small RNA Sequencing Read count Mapped length

17 Work to be done Phosphorylate the pools by kinase reactions Repeat TruSeq Small RNA sequencing Quantitative PCR TaqMan Array MicroRNA Card, Nadereh Jafari

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