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