Evaluation of MassCleave™ Technology For Diagnostic Mutation ...

Evaluation of MassCleave
MassCleave
Technology For Diagnostic
Mutation Detection
Chris Mattocks

PCR
T7-
MassCleave MassCleave
chemistry
Universal forward primer
with T7 promoter tag
C specific
cleavage product
Forward transcription
with T7 promoter
U specific
cleavage product
C specific
cleavage product
Reverse transcription
with T7 promoter
Mass spectrometry and comparison to predicted patterns
Universal reverse primer
with T7 promoter tag -T7
U specific
cleavage product
Ref: A strategy for the rapid discovery of disease markers using the MassARRAY system.
Rodi CP, Darnhofer-Patel B, Stanssens P, Zabeau M, van den Boom D. Biotechniques. 2002 Jun;Suppl:62-6, 68-9.

MassCleave MassCleave
Evaluation
�� Existing PCR designs
�� 12 fragments x 15 samples each + H 2O O control
�� Plate 1:
�� BRCA1 exon 11 - 6 fragments (B,C,G,J,K,L)
�� Plate 2:
�� hMLH1 – 5 fragments (exons ( exons 2,4,12,13 & 16)
�� MSH2 – exon 15 only
�� Sequenom provided primers
�� Amplifications carried out in NGRL
�� Analysis carried out by Sequenom

Mutation
type
Insertion
Deletion
Het. point
Hom. point
Total
Seq.
results
5
32
34
60
131
BRCA1 Results
flagged
4
26
34
60
126
Mutations identified with MassCLEAVE
(SNP discovery software aided analysis)
missed
1*
6*
0
0
7*
False
+ve
7
1
0
0
8+1 #
position
0
15
20
45
80
nature
*retrospective inspection of spectra clearly displays the mutation identifying signals
#one unclassified variant was called in a normal sample.
2
7
34
60
103
Zygosity
hom/het
3
26
34
60
123

Mutation
type
Insertion
Deletion
Het. point
Hom. point
Total
hMLH1 &hMSH2 Results
Seq.
results
5
12
25+1 #
4
46+1 #
flagged
4
10
25+1 #
4
44+1 #
Mutations identified with MassCLEAVE
(SNP discovery software aided analysis)
missed
0
2*
0
0
2*
False
+ve
0
0
0
0
0
position
4
1
25+1 #
4
34+1 #
nature
4
1
25
4
34
Zygosity
hom/het
5
8
25+1 #
3
41+1 #
*retrospective inspection of spectra clearly displays the mutation identifying signals.
#two base pair substitution, AA>GC

Rxn
TR
TR
CR
CR
mass
4918
5247
1689
2018
Theoretical cleavage
Relative
frequency
in normal
1
0
1
0
Relative
frequency
in mutant
0.5
0.5
0.5
0.5
GAAAACGGAGCAAAT
GAAAACAGGAGCAAAT
GGAGC
AGGAGC
Sequence

Frameshifts not flagged
�� 3819 del GTAAA base 154 to 158 fragment K - 3/3 missed
�� Only 1 additional signal (within normal analysis range)
�� V. Low potential score
�� Software not currently designed to detect 5 bp deletions
Rxn
TF
CF
CF
CR
mass
1658
6747
5110
1566
Relative
frequency
in normal
1
1
0
2
Relative
frequency
in mutant
0.5
0.5
0.5
1.5
AAAGT
TTGTTATTTGGTAAAGTAAAC
TTGTTATTTGGTAAAC
TTTAC
Sequence

3819 del GTAAA C-froward C froward

Frameshifts not flagged
�� 4184 del TCAA base 231-234 231 234 fragment L – 2/9 missed
�� Close proximity of 2 nd mutation
�� Only T-forward T forward gives unambiguous signals
�� Both amplicons failed/poor quality in T-forward T forward
�� T rich fragment in C-reverse C reverse
Rxn
TF
TF
CF
CF
CF
CR
CR
mass
5520
5247
2990
5210
6913*
3761
2479
Relative
frequency
in normal
1
0
1
1
0
1
0
Relative
frequency
in mutant
0.5
0.5
0.5
0.5
0.5
0.5
0.5
CAAGAAGAACAAAGCAT
AAGAAGAACAAAGCAT
AAGAAGAAC
TTGGAAGAAAATAATC
TTGGAAGAAAATAAGAAGAAC
TTGATTATTTTC
TTATTTTC
Sequence

Frameshifts Misscalled
�� 2731 ins T base 294 fragment G – miss-called miss called as 2731
C>T hom
�� Can only be discriminated by T-reverse T reverse and C-reverse C reverse
�� Presence second mutation
�� De-convoluted De convoluted in retrospect

2731 ins T T-reverse T reverse

Frameshifts Misscalled
�� 3450 del CAAG base 154 to 157 fragment J
�� Only T-forward T forward informative
�� Confounding noise (also seen in another sample)
�� Indicator seen in retrospect

3450 del CAAG T-forward T forward

Point mutations miss-located
miss located
�� 14 hets (3 unique)
�� 11 in fragments with 3 mutations
�� 2 in fragments with 2 mutations
�� 1 in fragment with 1 mutation
�� 15 Hom (1 mutation – 15/15 cases)
�� One of calls correct

Summary
�� Design non-optimal non optimal
�� All point mutations flagged and called but
position occasionally ambiguous
�� 32/37 frameshifts flagged – only 2 unique
mutations missed
�� 2 frameshifts misscalled - would need
confirmatory sequencing regardless

Conclusions
�� MassCleave MassCleave
provides:
�� Fast sample turn around
�� Very low false positive rate
�� Very promising comparative re-sequencing re sequencing method
for diagnostic screening
�� But:
�� More stringent (objective) quality criteria plus
extension of data analysis range needed

�� Larger study
Further work
�� Optimal design
�� Reference mutation controls
�� Defined quality criteria
�� Cost analysis
�� Software development
�� Mulitple alignments?
�� Frameshifts? Frameshifts
�� Work in progress by Sequenom

Acknowledgements
NGRL (Wessex)
Helen White
Niels Storm
Susan Muller

Preliminary report
available at http://www.ngrl.co.uk/Wessex/maldi_tof.htm
http:// www.ngrl.co.uk/Wessex/maldi_tof.htm