Exploring of microrna markers for body fluid identification using NGS

Transcription

1 Exploring of microrna markers for body fluid identification using NGS Zheng Wang, Yiping Hou Institute of Forensic Medicine Sichuan University, China Barcelona May, 11, 2016

2 Outline Introduction of Institute of Forensic Medicine (IFM) MicroRNA background Current methods for microrna profiling Powerful approach-ngs for small RNA sequencing

3 Outline Introduction of Institute of Forensic Medicine (IFM) MicroRNA background Current methods for microrna profiling Powerful approach-ngs for small RNA sequencing

4 Introduction Chengdu Land of Abundance 2300 years of city history

5 Introduction Pandas Hot pots

6 Introduction Institute of Forensic Medicine (IFM) Dean: Prof. Yiping Hou National Key Discipline of Forensic Medicine Offers M.S. degrees Offers Ph.D. degrees Receive the post-doctor About 25 graduate students each year Over 8000 forensic cases every year

7 Introduction Textbooks applied to the major of forensic science for national higher education in China All of them were edited by IFM

8 Introduction English Website:

9 Outline Introduction of Institute of Forensic Medicine (IFM) MicroRNA background Current methods for microrna profiling Powerful approach-ngs for small RNA sequencing

10 MicroRNA Biogenesis Pri-microRNA Rnase Ⅲ-Drosha Pre-microRNA Rnase Ⅲ-Dicer MicroRNA duplex Ago2 Mature microrna Cleavage Repression Deadenylation

11 MicroRNA Database mirbase

12 Main Characteristics Small RNA molecule: ~22 nucleotides in length Endogenous noncodingrna molecules Discovered in C.elegans (Lee et al., 1993) Essential regulative function in a large number of physiological contexts > 2500 mature micrornas already identified in H.sapiens Most highly evolutionarily conserved Highly specific and regulated expression pattern

13 However RNA was and is notorious for its rapid decay. Unfavorable environmental factors Stability remains one of the most important requirements of forensic markers.

14 Stability Lab conditions (1 year) Artificial light exposure Limited day light exposure Room temperature Room humidity It is remarkable that not only mirnas could be easily detected in experimentally aged body fluid samples but also that the absolute levels do not seem to be diminished in old samples. Zubakov D, et al. 2010, Int. J. Legal Med, 124:

15 Stability Lab conditions (1 month) Cq: unchanged Wang Z, et al Forensic Sci Int Genet, 7:

16 Stability FFPE samples Hui AB, et al. 2009, Lab Invest, 89(5):

17 Stability Leite KR, et al. 2011, Urol. Oncol, 19:

18 Outline Introduction of Institute of Forensic Medicine (IFM) MicroRNA background Current methods for microrna profiling Powerful approach-ngs for small RNA sequencing

19 Detection challenges ~22 nt length: insufficient for annealing to traditional primers Represent a small fraction (~0.01%) of the total RNA mass Lack a common sequence that can be used for selective enrichment MicroRNAs within a family: differ by as little as a single nucleotide Variance in GC content leads to a wide variance in Tm Sequence length variability Pritchard CC, et al. 2012, Nat Rev Genet. 13(5):

20 MicroRNA-profiling methods Conventional methods Microarray screening qpcr-array (TaqMan Array Human MicroRNA Cards) Real Time PCR validation Real Time-PCR TaqMan assays

21 Microarray Advantages Widely used in microrna profiling Fairly low-cost Best used for comparing relative abundance of specific mirnas between two states Disadvantages Fails to show a good inter-platform concordance Restricted linear range of quantification Imperfect specificity for microrna that are closely related in sequence Typically lower specificity than qrt-pcr or RNA sequencing Pritchard CC, et al. 2012, Nat Rev Genet. 13(5):

22 Real Time PCR Stem-loop primer: Reverse transcription of microrna to cdna TaqMan probe: Real-time monitoring of reaction product accumulation

23 Real Time PCR Advantages Sensitive and specific Easy to operate Can be used for absolute quantification Disadvantages Medium-throughput Cannot detect targets simultaneously Pritchard CC, et al. 2012, Nat Rev Genet. 13(5):

24 Our group: before Microarray screening and qpcr validation Real-Time PCR System

25 Our previous studies The model for data analysis of microrna expression levels Sample normalization Normalizer normalization Wang Z, et al. 2012, Forensic Sci Int Genet. 6(3):

26 Our previous studies Microarray screening and real time PCR validation Venous Blood: mir-486, mir-16 Semen: mir-888, mir-891a Menstrual Blood: mir-214 Saliva: mir205 + mir-891a Vaginal Secretions: mir-200c + mir mir-16 Wang Z, et al. 2013, Forensic Sci Int Genet. 7(1): Wang Z, et al. 2015, J Forensic Sci. 60(3):

27 MicroRNAs in human body fluids Silva SS, et al. 2015, Forensic Sci Int Genet. 14:1-10.

28 MicroRNA markers Groups Hanson et al. Zubakov et al. Courts et al. Park et al. Our group Venous blood mir-451 mir-16 mir-20a mir- 106a mir-185 mir-144 mir-126 mir-150 mir-451 mir-484 mir-182 mir-16 mir-486 Saliva mir-658 mir-205 mir-205 mir-200c mir-203 mir-223 mir-145 Semen mir-135b mir-10b mir-135a mir-10a mir- 507 mir-943 mir-891a mir-2392 mir-3197 mir-891a mir-888 Vaginal secretions mir-124a mir-372 mir-1260b mir-654 Menstrual blood mir-451 mir-412 mir-214 Wang Z, et al. 2016, Forensic Sci Int Genet. 20:

29 MicroRNA markers Not independently evaluated Few agreements among them Limited number Measuring the quantity of micrornas at the genome-wide level is an important step to search more specific micrornas for body fluid identification.

30 Outline Introduction of Institute of Forensic Medicine (IFM) MicroRNA background Current methods for microrna profiling Powerful approach-ngs for small RNA sequencing

31 NGS Next Generation Sequencing Also termed massively parallel sequencing (MPS) The capability to sequence many targeted regions Multiple samples simultaneously High coverage

32 NGS Next Generation Sequencing Also termed massively parallel sequencing (MPS) The capability to sequence many targeted regions Multiple samples simultaneously High coverage For microrna sequencing Allows both the detection of expression pattern and microrna sequences

33 Ion Personal Genome Machine Semiconductor Sequencing OneTouch ES OneTouch 2 Instrument Ion PGM

34 Our group: Since Microarray screening and qpcr validation Real-Time PCR System Ion PGM System

35 Workflow Sample Preparation MiRDong Small RNA library construction Torrent Suite v4.2 Template preparation Ion PGM sequencing

36 Sample Preparation 5 blood samples and 5 saliva samples RNA isolated by mirvana TM mirna Isolation kit Small RNA enriched by the Magnetic Bead Cleanup Module mirvana mirna Isolation Kit Magnetic Bead Cleanup Module

37 Library construction Hybridize and ligate the RNA, Perform reverse transcription Purify and size-select the cdna, Amplify the cdna Purify and size-select the amplified DNA Assess the yield and size distribution of the amplified DNA Pool barcoded small RNA libraries Ion Total RNA-Seq Kit Determine the library dilution required for template preparation

38 Typical size distribution

39 Sequence data analysis Criteria for micrornas calling 2588 human mature mirna sequences (mirbase v21) as alignment reference Perfect matching required: no longer, no shorter and no mismatch Sequence reads 100 counts MicroRNA frequency

40 Sequence data analysis Criteria for micrornas calling 2588 human mature mirna sequences (mirbase v21) as alignment reference Perfect matching required: no longer, no shorter and no mismatch Sequence reads 100 counts MicroRNA frequency micrornas in blood 143 micrornas in saliva

41 Cluster analysis Samples belonging to the same body fluid tend to cluster together Blood and saliva samples display distinct microrna expression signatures

42 Top 30 The top 30 high-expressed micrornas

43 Top 30 The top 30 high-expressed micrornas

44 Selection of micrornas The most informative microrna biomarkers Absolute expression levels in target body fluid The fold-change of differential expression between body fluids Preference given to micrornas are abundant in the target body fluids, but only minimally or not expressed in another body fluid.

45 Candidate marker

46 mirdong: Perl-based tool For microrna distribution designations Yandong Cao

47 Conclusions We used the Ion PGM TM System to profile the microrna distribution in blood and saliva. We present a reliable microrna workflow solution based on Ion PGM System. mirdong was developed for characterization of microrna profiling. 25 micrornas were proposed to distinguish between blood and saliva

48 Paper Wang Z, et al. 2016, Forensic Sci Int Genet. 20:

49 Further Other body fluids Semen: completed Menstrual blood and vaginal secretions: work in progress Skin cells Validating proposed microrna biomarkers Standardization of the procedure Simplifying analysis process

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52 TaqMan Probes, TaqMan assays, 3130 Genetic Analyzer, Ion PGM System and MirVana mirna Isolation Kits are For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. When used for purposes other than Human Identification the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures. Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration