SEQUENCIAMENTO DE TERCEIRA GERAÇÃO

1. miRNA Seq
Dr. Sikandar Hayat Khan
MBBS(Pak)
FCPS Chemical pathology (Pak)
PgD Diabetes & Endocrinology (UK)
MSc Cancer, Molecular Pathology & Genomics (UK)
Medicine joining hands with
bioinformatics: Emerging role of
miRNA in medical sciences

2. Sequence
Discussion context
Lab to informatics to clinics
Historical background
What is miRNA?
miRNA biogenesis
Role in medical sciences
miRNA measurement methods
miRNA extraction
Next phase
miRNA sequencing experience
miRNA sampling, study overview &
sample extraction
cDNA Library preparation
Sequencing
Biostatistics & bioinformatics
Results overview & miRNA
methodology biases
End note

3. Lab to informatics to clinics
• Bioinformatics is an
interdisciplinary field that
develops methods &
software tools for
understanding biological
data
• Bioinformatics combines
biology, computer science,
informatics, mathematics
and statistics to analyze &
interpret biological data
• Bioinformatics for in silico
analyses of biological
queries

4. Lab to informatics to clinics
-----Out of medic’s scope to interpret???

5. Lab to informatics to clinics
-----the Analytic Specialist

6. Lab to informatics to clinics
-----the growing role of AI (Pros & Cons)

7. Historical perspective

8. miRNA
• Small (22 nt)
• Non-coding RNA molecule
• Found in plants, animals & some viruses
• Functions:
• mRNA silencing
• Post-transcriptional regulation of gene expression
• miRNAs function via base-pairing with
complementary sequences within mRNA
molecules
• miRNA breaks down the mRNA by:
Cleavage of the mRNA strand into two pieces
Destabilization of the mRNA through shortening of
its poly(A) tail
Less efficient translation of the mRNA into
proteins by ribosomes
Human genome may
encode over 1000
miRNAs, which may
target about 60% of
mammalian genes

9. What is miRNA?
Available at:- https://www.slideshare.net/adnandinmohammed/01adnan. Retrieved on:-01-Jan-2020

10. miRNA biogenesis
passenger strand (miR*)
Guide strand (miR)

11. Diagnostics
Available at:- http://www.eurekaselect.com/154912/article
Retrieved on:-11-Jan-2020

12. Diagnostics
Available at:- https://www.frontiersin.org/files/Articles/116443/fcell-02-00061-r2/image_m/fcell-02-00061-g006.jpg.
Retrieved on:-13-Jan-2020

13. Therapeutics
Available at:- http://www.cantechletter.com/wp-content/uploads/2012/05/rnai-interference.jpg.
Retrieved on:-13-Jan-2020
RNA interference
RNA interference (RNAi) is a
biological process in which
RNA molecules inhibit gene
expression or translation, by
neutralizing targeted mRNA
molecules

14. Therapeutics
Available at:-.
Retrieved on:-13-Jan-2020
The FDA recently approved the first drug based on RNA
interference (RNAi) in 2018 for treating hereditary
transthyretin-mediated amyloidosis

15. miRNA measurement methods
Available at:- https://i.ytimg.com/vi/ZsiaHhHlwv8/hqdefault.jpg
Retrieved on:-13-Jan-2020
• Disease depiction vs Response (Acute phase
biomarker)
• Precise measurement of intracellular miRNA
expression is possible but can be challenging,
especially in the context of specialized tissue in vivo
• Sensitive to pre-processing & post-processing factors
• Accurate measurement of extracellular miRNA
presents other obstacles:
Low concentrations
Sources of intracellular miRNA that
contaminate RNA extraction
Methodology wise cost

16. miRNA measurement methods
Technique When to use Benefits Drawbacks
qPCR •Small scale
experiments (1–2
samples or miRNAs)
•Established protocols
•High sensitivity and specificity
•Labor intensive to scale
•Requires quality miRNA
annotation
miRNA arrays •Larger studies (has
been used for up to
900 samples)
•Established protocols
•Purpose built analysis tools
•Least quantitative
•Requires quality miRNA
annotation
miRNA-seq •Discovery phase
research
•Whole genome analysis
•Single base resolution
•Does not require miRNA
annotation
•Less sensitive than qPCR
•Requires most input material
•Good degree of technical and
bioinformatics skill necessary
Multiplex
miRNA
profiling
•Multiplex studies
(68 miRNAs, has
previously been
used for up to 600
samples)
•High sensitivity and specificity
•Straightforward data analysis
•Can be used on crude biofluids
•Requires standard lab equipment
•Not ideal for small scale
experiments of 1–2 samples
•Requires quality miRNA
annotation
nCounter
technology
•Under research
phase for miRNA
•No cDNA needed
•Faster
•Simpler
•Will be available to allow easy
multiplexing of many miRNA
•New technology
•Equipment not readily
available
•Needs replication at wider
scale

17. miRNA arrays technique
https://www2.vet.cornell.edu/sites/default/files/miRNAprofiling.png
miRNA profiling using microarray in different classes of BPH/5 mouse
feto-placental units

18. miRNA arrays technique
https://www2.vet.cornell.edu/sites/default/files/miRNAprofiling.png
Venn diagram showing
differentially expressed
miRNAs in BPH/5
placentas
Hierarchical clustering:
showing differential miRNA
expression in several
cardiovascular regulatory nuclei
of moues brain
Representative microarray
chip showing dysreguated
miRNA expression in BPH/5
mouse placenta

19. miRNA measurement methods
Available at:- https://www.mdpi.com/cells/cells-07-00219/article_deploy/html/images/cells-07-00219-g001-550.jpg. Retrieved on 17-Jan-2020.

20. Choosing miRNA Profiling Platform

21. Getting sample for miRNA
miRNA samples: MicroRNAs can be extracted from variety of specimens:
• Plasma/serum
• Cells in culture
• Fresh tissue/tumor
• Fixed tissue/tumors
Methods for purifying miRNA populations:
• Size purification by gel electrophoresis
• AGO2 immunoprecipitation (AGO2-IP)
 With crosslinking (CLIP)
 Without UV crosslinking (CLIP)
• Laser capture microdissection (LCM) can be used to purify material from
fresh-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections

22. miRNA Seq

23. Overall miRNA analytical plan
BLOOD SAMPLES STORAGE DONE AT -80oC
Sequencing was done in 3 batches of 6 DNA chips in replicates with
each run taking 7-10 days for:
Statistical analysis & bioinformatics
Quality control steps

24. Pre-extraction & sample processing
PRE-EXTRACTION
WORK UP
Samples allowed an
overnight stay (20-
25oC)
Avoiding RNases
contamination as per
guideline
QIAcube instrument
start up initiated
BM1 to BM4 & BR5
elution buffers
prepared
PROCESSING PAXgene
BLOOD RNA TUBE
PAXgene RNA tube was
centrifuged x 10-min
Supernatant was removed
Rnase-free water added
Tube was closed with fresh
secondary BD Hemograd
closure
Pellet was vortexed for
dissolving in solution
.
. PAXgene PROCESSING
BEFORE LOADING
Centrifugation again (10-
min), & supernatant
removal
350ul BM1 buffer (re-
suspension) added &
vortexed dissolved
Sample then pipetted into
2ml processing tube
Processing tubes loaded
on QIAcube shaker

25. PAXgene Blood miRNA product
Ready to use RNA> 18 nt (Including miRNA)
PAXgene® Blood miRNA kit hand book 2015. Available at: -
https://www.preanalytix.com/sites/default/files/handbooks/HB-0198-
003_1099367_HB_PAX_Blood_miRNA_1215_0.pdf. Retrieved on: 20-May-2018.
miRNA
extraction

26. miRNA QC CHECK
Methods can be used to assess miRNA
quality after extraction:
Spectrophotometry
Automated capillary electrophoresis
with the Bioanalyzer or Experion
AND/OR
Determining expression of
housekeeping miRNAs
For serum and plasma, which usually
have total RNA yields too low to
accurately quantify, determining the
recovery of spiked-in oligos can be a
useful surrogate

27. cDNA library preparation
Automated workflow
Minimal hands-on time
The Ion Chef System
fully automates:
 Library preparation
 Template
preparation
 Produces
sequencing-ready
Ion Torrent
semiconductor
chips

28. cDNA library
cDNA library is a combination of cloned cDNA
(complementary DNA) fragments inserted into a
collection of host cells, of which constitute some
portion of the transcriptome of the organism
and are stored as a "library"
cDNA is produced from fully transcribed mRNA
found in the nucleus and therefore contains only
the expressed genes of an organism

29. cDNA library

30. miRNA sequencing
• Following template
preparation on “Ion
Cheff”, miRNA
sequencing was
carried out on Ion
Proton sequencer as
per guidelines This
techniques relies upon
detection of H+ ions
once DNA sequences
polymerize
Ion PI™ Hi-Q™ Sequencing 200 Kit User Guide. Available at:
https://tools.thermofisher.com/content/sfs/manuals/MAN0010947_Ion_PI_HiQ_Seq_200_Kit_UG.pdf. Retrieved on: 07-June-2018

31. Bioinformatics

32. miRNA counts & fold change
• Finally the miRNA counts from replicates were
added and normalized by multiplying the individual
miRNA count with sum of all 1870 counts as:
1000000 x (ID3) / SUM(ID3:ID1872) in Excel document
(ID3 excel cell is one miRNA count)
• Fold change was calculated for each miRNA by
dividing cancer count/age-matched control counts
• Log2 fold change was calculated by using Excel
function

33. Various sequencing platforms
Adopted from: https://image.slidesharecdn.com/ngsoverviewparti-min-130401133728-phpapp02/95/a-comparison-of-ngs-
platforms-7-638.jpg?cb=1364823507. Retrieved on 30-July 2018

34. Sequencing variables
Pertinent here is to define the variables which can cause
sequencing variability to result in inter and intra-
instrument miRNAs data differences
18/04/2021 46
miRNA
data
variability
RNA
extraction
procedure
Inherent
to
miRNA
Platform
related
Bioinformatics
NGS platform selection affect
results
• Life Technologies Ion Torrent
• 454 Life Sciences (Roche)
• Pacific Biosciences
• Nanopore DNA technology
• Helicos BioSciences
platforms
Ref: Bahassi M et al. Next-generation sequencing
technologies: breaking the sound barrier of human
genetics. Mutagenesis. 2014
Variability can result from the
unique proportion of miRNAs
like
• Length differences
• Sequene heterogeneity
• GC contents
• Possibly SNPs
Ref: Wang Z et al. Characterization of microRNA
expression profiles in blood and saliva using the Ion
Personal Genome Machine(®) System (Ion PGM™
System). Forensic Sci Int Genet. 2016
RNA extraction technologies
results
• Differences in RNA extraction
procedure
• Separate RNA work area
• Enrichment needs
• Associated laboratory
competencies
• RNAse contamination
Ref: Bergallo M et al, Comparison of Two Available
RNA Extraction Protocols for microRNA
Amplification in Serum Samples. J Clin Lab Anal.
2016

35. End note
• miRNA diagnostics can provide valuable insight into
disease pathology & related therapeutic approaches like
RNAi
• Appropriate methodology selection for miRNA
measurement is mandatory
• Aspects related to miRNA behavior, miRNA extraction &
purification and associated QC need to be applied
• miRNA Seq can provide a wholesome approach for
identifying novel miRNA as disease biomarkers &
prediction
• miRNA data interpretation needs bioinformatics knowledge
• Bioinformatics need to be a part of molecular pathology

36. End note
• Molecular diagnostics, gene therapy & molecular
therapeutics will replace conventional medicine for good
in future
• Unmet need is there to develop & incorporate latest
molecular pathology services in labs
• Bioinformatics with its human & IT resource with links to
validated genome browsers like “UCSC” genome
browsers, COSMIC & Type2 DIABTES KNOWLEDGE
PORTAL is pivotal to our future in healthcare
• Medical doctors, biotechnologists & bioinformaticians
need to be aware of CRISPR technologies, bio banking &
emerging nano precise molecular pathology tools

37. Lab to informatics to clinics
-----AI & IT will take over many jobs

38. Thanks

39. Khan S H. (2019). TOWARDS SYNTHETIC GENOMICS: WHAT NEXT TO COME AFTER GENOME EDITING?.
Pakistan Armed Forces Medical Journal, 69(5), 931-33. Retrieved from https://pafmj.org/index.php/PAFMJ/article/view/3387
Khan S H. JGM CC-license DOI : 10.14302/issn.2572-5424.jgm-19-3024 Vol-1 Issue 3 Pg. no.- 34 Molecular and
Metabolic Pathogenesis of Familial Combined Hyperlipidemia and Association with Metabolic Syndrome.
Khan S H.Review: Genome-Editing Technologies: Concept, Pros, and Cons of Various Genome-Editing
Techniques and Bioethical Concerns for Clinical Application. Molecular Therapy: Nucleic Acids Vol. 16 June 2019
Khan S H, Sarwar U. Newer molecular insights into type-2 diabetes mellitus. Accepted for publication in
JPMA. DOI: http://doi.org/10.5455/JPMA.20855.
Khan S H. JOM CC-license DOI : 10.14302/issn.2574-450X.jom-19-3001 Vol-1 Issue 3 Pg. no.- 1 JOURNAL OF
OBESITY MANAGEMENT ISSN NO: 2574-450X Short communication Type-2 Diabetes and Gene Therapy: The
Promise of CRISPR Gene Therapy in type-2 Diabetes Mellitus.
Khan SH. EDITORIAL: ENTERING THE ERA OF MOLECULAR MEDICINE: ARE WE PREPARED? Pak Armed
Forces Med J 2018; 68 (5): 1056-58
Khan SH. Blood miRNA in type-2 Diabetes Mellitus? Possible futuristic molecular biomarkers.
Being submitted in “Molecular Therapy - Methods & Clinical Development”
Khan SH. Recent advancement and innovations in CRISPR/Cas and CRISPR related technologies: A
review. Submitted for publication in “Molecular Therapy - Methods & Clinical Development “
Publications

40. Q &A

41. Supplementary data

42. miRNA profiling

43. NGS Platforms
Advantages Disadvantages Assay/Platform Vendor
RNA*
req
material
costs
/sample** Reference
• High accuracy and
sensitvity.
• Can detect novel
miRNAs
• Significant
computationa
l support
needed for
data analysis.
• Cannot be
used to
determine
absolute
quantification
.
High Throughput Next Generation
Sequencing Platforms
+++
$$$
HiSeq™ 2000 (Genome
Analyzer IIX)
Illumina
Kato, et al 2011
SOLiD™ ABI Ramsingh, et al 2010,
Schulte, et al 2010
GS FLX+ (454) Roche Wyman, et al 2009;
Rajagopalan et al 2006,
Soares et al 2012
Smaller Scale Next Generation
Sequencing Platforms
Ion Torrent™ Invitrogen
MiSeq™ Illumina
GS Junior (454) Roche
• Amplification not
required.
• Potential to
determine absolute
quantification
• Expensive
• Currently less
sensitive
Single Molecule Sequencing
Technologies
$$$$
tSMS™ Helicos Kapranov, et al 2010
SMRT™
Pacific
Biosciences

44. qRT-PCR
Advantages Disadvantages Assay/Platform Vendor
RNA*
required
material costs
per sample** reference
• Established
method,
sensitive and
specific.
• Easily adapted
to existing
real-time PCR
workflow.
• Can be used
to determine
absolute
quantification
.
• Customizable
Cannot
identify novel
miRNA
(including in
less
frequently
studied
species)
TaqMan® individual
Assays
ABI
+ – ++ $ – $$
Chen, et al
2005, PMID
21642990
miRcury LNA™
qPCR Primers
Exiqon
TaqMan®
OpenArray
ABI
TaqMan® TLDA
Microfluidics Card
ABI
Junker, et al
2009
Biomark™ HD
System
Fluidigm
Petriv, et al
2010
SmartChip Human
MicroRNA
Wafergen
Keller, et al
2011
miScript miRNA
PCR Array
SABiosciences/
Qiagen

45. MicroRNA Microarray
Advantages Disadvantages Assay/Platform Vendor RNA* required
material costs
per sample** Ref
• Established
method.
• Easily adapted
to existing
microarray
workflow.
• Lower specificity
than RNAseq.
• Cannot be used to
determine
absolute
quantification.
Geniom Biochip
miRNA
CBC (febit)
++ $
Keller, et
al 2011
miRCURY LNA™
microRNA Array
Exiqon
μParaFlo™ Biochip
Array
LC Biosciences
MicroRNA Microarray Agilent
GeneChip® miRNA
Array
Affymetrix
OneArray®
Phalanx
Biotech
Sentrix® Array Matrix
and BeadChips
Illumina
GenoExplorer™ Genosensor

46. miRNA related nomenclature
Naming
Convention
Meaning Example(s)
3 letter prefix
Species identification. This prefix is sometimes dropped in some
journals, with the species under consideration mentioned in the
adjoining text.
hsa (human)
dme (fruit fly)
pri-mir or pre-mir (not
lower case “r”)
Used to indicate primary transcript of miRNA (pri-mir) or
precursor form after Drosha processing (pri-mir)
pri-mir-16, pre-mir-16
miR Mature miRNA hsa-miR-16
−3p or −5p
Mature miRNA originating from the 3′ or 5′ end of the pre-
miRNA, respectively
hsa-miR-142-3p
hsa-miR-142-5p
a or b
Closely related miRNAs that are related in sequence and
evolutionary origin
hsa-miR-20a
hsa-miR-20b
−1, or −2
Identical mature miRNA sequences that originate from different
genomic loci
hsa-miR-16-1
hsa-miR-16-2
miR* (star)
‘Passenger strand’ found at lower concentration, usually
degraded (retired in miRBase 17)
hsa-miR-9*

47. miRNA sources
Sample type Applications miRNA Yield* Considerations
Cell lines Gene Regulation +++ High quality miRNA usually
Fluorescence activated cell
sorted cells Translational, Basic Science ++
Lower yield, less
heterogeneity
Fresh tissue (e.g., tumor) Translational Research +++ Cell heterogeneity
Formalin-fixed, paraffin-
embedded tissue
Clinical, Translational
Research ++
More reliable than mRNA as
an analyte in FFPE
Laser-capture
microdissected tissue Translational Research +
Less heterogeneity, but lower
yeild, never completely pure
Plasma, Serum Biomarkers +
RNAses, low yield, typically
cannot evaluate quality or
quantity
Urine Biomarkers +
Can evaluate cell pellet vs.
supernatant

48. Approaches to miRNA Profiling

49. Approaches to miRNA Profiling
(A)qRT-PCR. In TaqMan qRT-PCR the reverse transcription (RT) reactions use stem loop primers
specific to the 3′ end of the miRNA for specificity (A, top left). Amplicons are generated using an
miRNA-specific forward primer, where DNA polymerase proceeds along template, the TaqMan probe
is hydrolyzed so the quencher is freed from fluorescent dye, resulting in light emission (A, top
middle). In SYBR green-based qRT-PCR miRNA is polyadenylated at the 3′ end and Oligo d(T) used
as an RT-primer (A, bottom left). An miRNA-specific forward primer and oligo d(T) enable PCR
amplification with dsDNA-intercalating SYBR green dye as the detector (A, bottom middle). Both
TaqMan and SYBR green-based qRT-PCR are available in medium format “array” (A, right).
(B)miRNA Microarray. DNA-based capture probes (which may or may not incorporate LNA-modified
bases) are used to capture fluorescently-tagged miRNAs, followed by scanning of slides and
quantification of fluorescence. Several variations on this approach exist.
(C)RNA Sequencing. Current established RNA sequencing platforms begin with reverse transcription of
miRNA to a cDNA library. Adaptor ligation then allows the library to either be affixed to a solid phase
as in the Illumina platform or to beads for emulsion PCR as in the Roche and ABI platforms. For
details of RNA sequencing chemistry see Metzker 2010
(D)Nanostring NCounter: Two target-specific probes are designed for each miRNA of interest: a 3′
capture probe containing biotin to allow adsorbance to solid phase via strepavidin, and a second 5′
reporter probe with an individual color-coded sequence. No amplification or labeling is required with
this method.

50. • Precise measurement of
intracellular miRNA expression is
possible but can be challenging,
especially in the context of
specialized tissue niches in vivo.
• The accurate measurement of
extracellular miRNA presents
other obstacles stemming from
their low concentrations &
confounding sources of
intracellular miRNA that
contaminate RNA extraction
protocols

51. Available at:-
https://image.slidesharecdn.com/exiqonwebinar23feb2012finalpdf-13351798529928-phpapp02-120423062035-phpapp02/95/microrna-
discovery-and-biomarker-development-in-clinical-samples-25-728.jpg?cb=1335162082. Retrieved on: 19-Jan-2020.

52. miRNA Seq web browser
Available at:- https://i.ytimg.com/vi/ZsiaHhHlwv8/hqdefault.jpg
Retrieved on:-13-Jan-2020

53. Lab to informatics to clinics
-----Only team work can bring in real cure for the ailing

54. miRNA measurement methods
• Bioinformatics plays a big
part in identifying putative
miRNAs, they also need to be
experimentally verified in lab
• Vrious techniques developed
to overcome the challenges
of miRNA profiling
• We discuss popular methods
currently in use
Available at:- https://depositphotos.com/50021347/stock-photo-workplace-modern-laboratory-for-molecular.html.
Retrieved on:- 14-Jan-2020