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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
Outline
• Introduction of Institute of Forensic Medicine (IFM)
• MicroRNA background
• Current methods for microRNA profiling
• Powerful approach-NGS for small RNA sequencing
Outline
• Introduction of Institute of Forensic Medicine (IFM)
• MicroRNA background
• Current methods for microRNA profiling
• Powerful approach-NGS for small RNA sequencing
Introduction
Chengdu
“Land of Abundance”
2300 years of city history
Introduction
Hot pots
Pandas
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
Introduction
Textbooks applied to the major of forensic science
for national higher education in China
All of them were edited by IFM
Introduction
• English Website: http://www.legalmed.org/Eindex.html
Introduction
Outline
• Introduction of Institute of Forensic Medicine (IFM)
• MicroRNA background
• Current methods for microRNA profiling
• Powerful approach-NGS for small RNA sequencing
Mature microRNA
Pri-microRNA
Pre-microRNA
Rnase Ⅲ-Dicer
MicroRNA duplex
Rnase Ⅲ-Drosha
Ago2
Cleavage Repression Deadenylation
MicroRNA Biogenesis
MicroRNA Database
miRBase
http://www.mirbase.org
• 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
Main Characteristics
However
RNA was and is notorious for its rapid decay.
Unfavorable environmental factors
Stability remains one of the most important requirements of forensic markers.
• 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: 217–226.
Stability
Lab conditions
(1 year)
Lab conditions
(1 month)
∆Cq: unchanged
Wang Z, et al. 2013. Forensic Sci Int Genet, 7:116–123.
Stability
Stability
Hui AB, et al. 2009, Lab Invest, 89(5): 597-606.
FFPE samples
Leite KR, et al. 2011, Urol. Oncol, 19: 533–537.
Stability
Outline
• Introduction of Institute of Forensic Medicine (IFM)
• MicroRNA background
• Current methods for microRNA profiling
• Powerful approach-NGS for small RNA sequencing
• ~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
Detection challenges
Pritchard CC, et al. 2012, Nat Rev Genet. 13(5): 358-369.
MicroRNA-profiling methods
Conventional methods
Microarray screening
Real Time PCR validation
qPCR-array
(TaqMan Array Human MicroRNA Cards)
Real Time-PCR
TaqMan assays
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): 358-369.
Real Time PCR
• Stem-loop primer: Reverse transcription of microRNA to cDNA
• TaqMan probe: Real-time monitoring of reaction product accumulation
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): 358-369.
Real Time PCR
2014 2015
Real-Time PCR System
Our group: before 2015
Microarray screening and qPCR validation
2010
Our previous studies
Sample normalization Normalizer normalization
The model for data analysis of microRNA expression levels
Wang Z, et al. 2012, Forensic Sci Int Genet. 6(3): 419-423.
Our previous studies
Venous Blood: miR-486, miR-16
Semen: miR-888, miR-891a
Menstrual Blood: miR-214
Saliva: miR205 + miR-891a
Vaginal Secretions: miR-200c + miR-214 + miR-16
Microarray screening and real time PCR validation
Wang Z, et al. 2013, Forensic Sci Int Genet. 7(1): 116-123.
Wang Z, et al. 2015, J Forensic Sci. 60(3): 702-706.
MicroRNAs in human body fluids
Silva SS, et al. 2015, Forensic Sci Int Genet. 14:1-10.
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. 20:140-146.
MicroRNA markers
Measuring the quantity of microRNAs at the genome-wide level is an important
step to search more specific microRNAs for body fluid identification.
• Not independently evaluated
• Few agreements among them
• Limited number
MicroRNA markers
Outline
• Introduction of Institute of Forensic Medicine (IFM)
• MicroRNA background
• Current methods for microRNA profiling
• Powerful approach-NGS for small RNA sequencing
NGS
Next Generation Sequencing
• Also termed massively parallel sequencing (MPS)
• The capability to sequence many targeted regions
• Multiple samples simultaneously
• High coverage
NGS
Next Generation Sequencing
• Also termed massively parallel sequencing (MPS)
• The capability to sequence many targeted regions
• Multiple samples simultaneously
• High coverage
Allows both the detection of expression pattern and microRNA sequences
For microRNA sequencing
Semiconductor Sequencing
Ion Personal Genome Machine
OneTouch™ 2 InstrumentOneTouch ™ ES Ion PGM ™
2014 2015
Real-Time PCR System
Our group: Since 2015
2010
Ion PGM™ System
Microarray screening and qPCR validation
Sample Preparation
Small RNA library construction
Template preparation Ion PGM sequencing
Torrent Suite v4.2
Workflow
MiRDong
mirVana™ miRNA Isolation Kit
Sample Preparation
• 5 blood samples and 5 saliva samples
• RNA isolated by mirVanaTM miRNA Isolation kit
• Small RNA enriched by the Magnetic Bead Cleanup Module
Magnetic Bead Cleanup Module
Library construction
Ion Total RNA-Seq Kit
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
Determine the library dilution required for template preparation
Typical size distribution
Sequence data analysis
• 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 ≥ 0.0001
Criteria for microRNAs calling
Sequence data analysis
• 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 ≥ 0.0001
Criteria for microRNAs calling
79 microRNAs in blood
143 microRNAs in saliva
Cluster analysis
• Samples belonging to the same body
fluid tend to cluster together
• Blood and saliva samples display distinct
microRNA expression signatures
The top 30 high-expressed microRNAs
Top 30
The top 30 high-expressed microRNAs
Top 30
Selection of microRNAs
• Absolute expression levels in target body fluid
• The fold-change of differential expression between body fluids
The most informative microRNA biomarkers
Preference given to microRNAs are abundant in the target body fluids,
but only minimally or not expressed in another body fluid.
Candidate marker
miRDong: Perl-based tool
yandong.cao@analyses.cn
Yandong Cao
For microRNA distribution designations
• We used the Ion PGMTM 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
Conclusions
Paper
Wang Z, et al. 2016, Forensic Sci Int Genet. 20:140-146.
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
wangzhengtim@163.com
TaqMan Probes, TaqMan assays, 3130 Genetic Analyzer, Ion PGM System and MirVana miRNAIsolation 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
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