Emerging Technologies in Molecular Pathology
Edward C. Stack, Ph.D.
January 5, 2012
Molecular techniques can offer tremendous insight into disease processes. However, indiscriminate use of these techniques, without a clear understanding of underlying pathology, can lead to false assumptions.
• How pathology guides molecular analysis• Dissection methods and consequences
How pathology guides molecular analysis
How pathology guides molecular analysis
Dissection methods and consequences
Microdissection Macrodissection
Dissection methods and consequences
RNA Profiles
Robot Row in CMOP
Nucleic Acid Extraction
Fres
hFF
PE
• NanoString for limited transcriptome analysis• Affy ST microarrays for more complete transcriptome coverage• RNAseq for whole transcriptome coverage
• Fresh v. FFPE• RNA species differences• analysis issues and biocomputational evolution
Emerging tools in the molecular pathology armory
As new tools avail themselves, it is important to understand those currently used and how new assays, such as nanoString or RNAseq, compare to those currently considered the ‘gold’ standard, such as TaqMan qPCR.
Emerging tools in the molecular pathology armory
qPCR
A technical advancement over standard PCR, as developed by Mullis and Faloona.
PCR allows for exponential amplification of DNA sequences, when the genomic sequence at each end of the target is known.
Importantly, quantification (the q in qPCR) of expressed transcripts is not possible with standard PCR, which is more qualitative and at best, semi-quantitative. Why?
It is essential to understand standard PCR in order to make effective use of qPCR.
Emerging tools in the molecular pathology armory
qPCRStandard
PCRStrand Separation Primer Hybridization DNA synthesis
Emerging tools in the molecular pathology armory
qPCRThe RT portion – can be performed as
either a one- or two-step process
Emerging tools in the molecular pathology armory
qPCRUsing TaqMan Reagents (Applied Biosciences)
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr/taqman-and-sybr-green-chemistries.html
Emerging tools in the molecular pathology armory
qPCR
qPCR allows reactions to be characterized by the point in time during cycling when amplification of a PCR product achieves a fixed level of fluorescence.
In the initial PCR cycles (baseline), fluorescence is negligible. Software then calculates the Rn (normalized reporter), with an algorithm determining the point on the amplification plot at which the ∆Rn crosses the threshold. The cycle at which this occurs is defined as CT.
Emerging tools in the molecular pathology armory
qPCR In practice - interrogating miR 146a (with RNU48 snoRNA control).
Emerging tools in the molecular pathology armory
NanoString
This novel transcription analysis was developed within the last 5 years, and is proving to be an attractive alternative to qPCR for comprehensive, though still small, transcriptome studies.
Developed by Geiss and colleagues -
Emerging tools in the molecular pathology armory
NanoStringThrough use of a capture and reporter pairing, this assay is capable of
detecting ~16K individual transcripts. How it works:
From Geiss, et al (2008)
Emerging tools in the molecular pathology armory
NanoStringAnalysis of spiked-in controls provided demonstrates linearity and
reproducibility of the assay
From Geiss, et al (2008)
Emerging tools in the molecular pathology armory
NanoStringScatterplot analysis of normalized NanoString signal shows very tight R2. GEP
alterations also observed.
From Geiss, et al (2008)
Emerging tools in the molecular pathology armory
NanoStringComparison between NanoString and an Affy array demonstrated higher
transcript detection in the Nanostring platform.
From Geiss, et al (2008)
Emerging tools in the molecular pathology armory
NanoStringFurther comparisons of fold-changes between NanoString and an Affy array
demonstrated fairly good agreement (R2 = 0.788).
From Geiss, et al (2008)
For 202 transcripts significantly regulated in both the NanoString and Affy platforms, the two platforms demonstrated significant agreement (Correlation coefficient of 0.788). .
Emerging tools in the molecular pathology armory
NanoStringAdditional validation of detected fold changes between NanoString and Affy
using TaqMan revealed very high concordance (more so than Affy). Furthermore, using human reference RNA, the concordance between NanoString and TaqMan persists (R2 = 0.945). What does this mean?
From Geiss, et al (2008)
Emerging tools in the molecular pathology armory
NanoStringSo how well does it work on FFPE?
NanoString qPCR
Pearson Coefficient r = 0.59
Pearson Coefficient r = 0.90 Pearson Coefficient r = 0.50
Samples are FFPE oral carcinomas
Emerging tools in the molecular pathology armory
Affy ST microarray
The Affy 1.0 ST Array interrogates 28,869 genes using 764,885 unique probes (~40/gene), based on more than a 3’-based interrogation. Why might this be important in FFPE?
Total RNA
whole transcriptome amplification
cDNA
Figure courtesy of Neil Martin, MD
Emerging tools in the molecular pathology armory
Affy ST microarray How does the array work?
Emerging tools in the molecular pathology armory
Affy ST microarray Does the array work well in FFPE? • Pilot data from sample 1175.
Log2 ratios between paired fresh and FFPECorrelation trending correctly.
Emerging tools in the molecular pathology armory
Affy ST microarray Does the array work well in FFPE?
Normalized data clusters based on tissue type – squamous cell carcinoma or adenocarcinoma. Putative p63
signature (correlative and anti-correlative changes) between SCC and AC.
From Hall, et al, 2011
Emerging tools in the molecular pathology armory
Affy ST microarray
Post-assay considerations:
• Bioinformatics required for parsing of the data.
• Unlike the NanoString, calls of gene up/down regulation need to be verified by an independent method – typically TaqMan.
Emerging tools in the molecular pathology armory
RNAseq
Shorthand for R N A Sequencing.Developed in the late 1990’s by Cambridge scientists Shankar Balasubramanian and David Klenerman, in part through their work on fluoroescently labeled captured DNA molecules.
Allows for the sequencing of ALL RNA species, providing an unparalleled view of the entire transcriptome.
Emerging tools in the molecular pathology armory
RNAseqOverview.
Transcriptome reconstruction
Expression quantification
From Wang et al, 2009
FreshTissu
eCores
RNA from FreshTissu
e
RNA Isolation Typical RNA Profile from FFPE TruSeq Library Prep Blunt End°°
Adenylate°°
Ligate Adapter
°°Enrich Library
Typical TruSeq Library Profile from FFPE
1st Strand
2nd Strand
Emerging tools in the molecular pathology armory
RNAseqLibrary Construction – Fresh RNA
FFPE Tissu
eCores
RNA from FFPE Tissu
e
RNA Isolation Typical RNA Profile from FFPE TruSeq Library Prep Blunt End°°
Adenylate°°
Ligate Adapter
°°Enrich LibraryRNA
from FFPE
Tissue
1st Strand
2nd Strand
Emerging tools in the molecular pathology armory
RNAseqLibrary Construction – FFPE RNA
Typical TruSeq Library Profile from FFPE
Emerging tools in the molecular pathology armory
RNAseqData Generated – Fresh RNA
Reads / Aligned Reads
Chromosome Map
Emerging tools in the molecular pathology armory
RNAseqData Generated – Feasibility in FFPE RNA
Reads / Aligned / Not / Multi
Chromosome Map
Typical visualization of expression (PTEN) in replicate samples from FFPE blocks containing prostate cancer tissue.
While expression is not restricted to exons, there is a tight segregation of reads within identified gene regions, and a general absence of reads outside of identified transcripts.
Emerging tools in the molecular pathology armory
RNAseqData Visualized – Feasibility in FFPE RNA
0200
400600
8001000
12001400
16001800
20000
200400600800
100012001400160018002000
Gene FPKM Concordance
R1, FPKM
R2, F
PKM
Concordance over 10,000 gene transcripts detected by RNAseq in replicate FFPE PrCa samples. R2 = 0.9838
0 10 20 30 40 50 60 70 80 90 1000
20
40
60
80
100
Concordance over 7000 transcription starts sites (TSS) detected by RNAseq in replicate FFPE PrCa samples. R2 = 0.9989
TSS FPKM Concordance
R2, F
PKM
R1, FPKM
A Sample of PrCa-relevant genes detected include:MMP10, SMTN, ERG, TMPRSS2, BIRC5, UBE2C, CDKN3, VEGF, and TK1.
Emerging tools in the molecular pathology armory
RNAseqData Visualized – Feasibility in FFPE RNA
Emerging tools in the molecular pathology armory
RNAseqFeasibility in FFPE RNA
A fairly robust read depth via single end sequencing results in ~ 40% alignment to hg18 sufficient to support interrogation of the transcriptome.
Inter-assay reproducibility is demonstrated by high concordant replicates, for both gene transcripts and transition start site sequences, demonstrating high assay fidelity.
Lack of poly-A selection offers more transcriptome coverage, providing a more complete snapshot of the RNA environment, including mRNA, ncRNA, etc..
Does this mean RNAseq is ready for research?
Emerging tools in the molecular pathology armory
RNAseqIssues with FFPE RNA
Bioinformatic challenges include:
Data handling and storage (sequencing leads to massive data outputs).
Contig assembly and genomic alignment (we currently use TopHat, though there are others). Complex genomes though have problems with:
exon spanning sequences (junction library);sequence reads that map to multiple genomic locations (proportional assignment
based on neighboring unique sequences).
Cost versus coverage and depth – real world concerns.
Emerging tools in the molecular pathology armory
RNAseqIssues with FFPE RNA
Mapping aligned reads to UCSC Genome browser demonstrate marked differences between fresh and FFPE RNAseq samples in the housekeeping gene transcript ALDOA - aldolase A, fructose-bisphosphate.
From Dr. Svitlana Tyekucheva
Emerging tools in the molecular pathology armory
RNAseqIssues with FFPE RNA
A similar trend was observed when mapping aligned reads to UCSC Genome browser demonstrate marked differences between fresh and FFPE RNAseq samples in: the Pca specific gene transcript KLK3 - kallikrein-related peptidase 3 (or PSA); or the housekeeping gene transcript GAPDH - glyceraldehyde-3-phosphate dehydrogenase.
From Dr. Svitlana Tyekucheva
GAPDH
KLK3
Emerging tools in the molecular pathology armory
RNAseqIssues with FFPE RNA
A similar trend was observed when mapping aligned reads to UCSC Genome browser demonstrate marked differences between fresh and FFPE RNAseq samples in: the housekeeping gene transcript ACTNB- Actin B; or the housekeeping gene transcript RPL32- ribosomal protein L321.
While the apparent loss of aligned signal is less than ideal, the overall picture suggests that the development of novel bioinformatic approaches to FFPE RNAseq data will lead to a new era in transcriptome analyses.
From Dr. Svitlana Tyekucheva
RPL32
ACTNB
Emerging tools in the molecular pathology armory
RNAseqFFPE small RNA – miRRNAseq in practice!
Small RNAseq (smRNAseq), where the assay can detect miRNA snoRNA, scRNA, snRNA, as well at tRNA. Mt-tRNA, rRNA, introninc, exonic, and more…
Emerging tools in the molecular pathology armory
RNAseqFFPE small RNA – miRRNAseq in practice!
Unsupervised cluster analysis of miR expression calls between RCC and normal as assessed by RNAseq in both fresh and FFPE samples demonstrates the ability of each substrate to classify tumor from normal.
Notice anything unusual?
From Weng et al, 2010
Emerging tools in the molecular pathology armory
RNAseqFFPE small RNA – miRRNAseq in practice!
Correlational analysis of miR expression calls between RCC and normal as assessed by both RNAseq and microarray in fresh samples demonstrates high correlation of expression alterations (R=~0.8). Same is true for differentially expressed miR’s (R=0.77).
Data demonstrate practical suitability for large-scale smRNAseq.
From Weng et al, 2010
Emerging tools in the molecular pathology armory
RNAseqFFPE small RNA – miRRNAseq in practice!
Correlational analysis of miR expression calls between RCC and normal as assessed by RNAseq in fresh and FFPE samples also demonstrates high correlation of expression alterations (R=~0.96). Differentially expressed miR’s show similar concordance as before (R=0.79).
Anything unusual about this approach?
From Weng et al, 2010
Emerging tools in the molecular pathology armory
RNAseqFFPE small RNA – miRRNAseq in practice!
Specific miR expression levels by RNAseq, microarray, and qPCR in fresh samples
Does the data presented leave any impression?
From Weng et al, 2010
Emerging Technologies in Molecular PathologyReference List:
Geiss GK, et al.. (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317-325.
Hall JS, Leong HS, Armenoult LS, Newton GE, Valentine HR, Irlam JJ, Möller-Levet C, Sikand KA, Pepper SD, Miller CJ, West CM. (2011) Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples. Br J Cancer 104:971-981.
Mullis KB, Faloona FA. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335-350.
Osborne MA, Furey WS, Klenerman D, Balasubramanian S.(2000) Single-molecule analysis of DNA immobilized on microspheres. Anal Chem 72:3678-3681.
Reis PP, Waldron L, Goswami RS, Xu W, Xuan Y, Perez-Ordonez B, Gullane P, Irish J, Jurisica I, Kamel-Reid S. (2011) mRNA transcript quantification in archival samples using multiplexed, color-coded probes. BMC Biotechnol 11:46-56.
Weng L, Wu X, Gao H, Mu B, Li X, Wang JH, Guo C, Jin JM, Chen Z, Covarrubias M, Yuan YC, Weiss LM, Wu H. (2010) MicroRNA profiling of clear cell renal cell carcinoma by whole-genome small RNA deep sequencing of paired frozen and formalin-fixed, paraffin-embedded tissue specimens. J Pathol 222:41-51.
Wang Z, Gerstein M, Snyder M. (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57-63.