Measles and Rubella Viruses Molecular

Quality Control and Analysis Workshop

Institute Malbran
Buenos Aires, Argentina
11-13 April 2018

National Center for Immunization & Respiratory Diseases

Division of Viral Diseases
 Key Activities-Objectives
Day 1: Real time RT-qPCR for the detection of measles and rubella viruses
• Quality control of assay
• Monitoring controls: QMS
• Troubleshooting
Day 2: Sequence Analysis for genotyping measles and rubella viruses
• Quality control
• Troubleshooting
• Sequence analysis software options:
 Mega
 GeneStudio
 ReCall
Day 3: MeaNS, RubeNS, and GeneBank databases
• Submission
• Named strains
• GenBank tools
Sequence Analysis for genotyping measles
and rubella viruses

• Quality control
• Troubleshooting
• Sequence analysis software options:
 Mega
 GeneStudio
 ReCall
 Measles Genotyping
N P/C/V M F H L

seq window

1104 1131 1233 1686 1747

seq window
MeV216 MeV214

Primers Amplify 104 Copies of RNA Template

• Primers MeV214 and MeV216 are designed to amplify a
634 nucleotide region coding for the 3’ terminus of the
nucleoprotein (N) gene in a conventional RT-PCR
reaction.

• 11 different genotypes tested

• MV 214 and 216 are also used in sequencing reactions

 Measles Control RNA (MeV-N3in)
220 nt insert

genotyping PCR product

Real-time PCR product

• The control RNA (MeV-N3in) is a synthetic N gene RNA that

contains an insert to increase the size of the RT-PCR product (to
854 bp) compared to PCR products from patient specimens (634
bp). This reduces the risk of misinterpretation due to cross-
contamination.

• The synthetic RNA can be used for genotyping RT-PCR or real-time

diagnostic RT-PCR.
 Agarose Gel with Primers and Control

1200 nt
854 nt
800 nt
634 nt
500 nt
300 nt

1 2 3 4
Lane 1: Positive sample
Lane 2: Negative control
Lane 3: Positive control with insert
Lane 4: MW marker
 Rubella Genotyping

• Rubella sequence window is 739 nucleotides in length

• RT-PCR amplicon needs to be larger (e.g. 945 nt) to
allow for primer binding sites and low quality sequence
close to the primer sites
• In general, the larger the amplicon, the higher the
minimum copy number required for template production
• This is NOT the same gene used for detection in the

real time RT-qPCR assay

 Rubella Virus Genotyping Using
2 Overlapping Amplicons
E1 gene
coding region
Molecular Window (739-nt)

Sensitivity
Single amplicon
(945 bp) 103 copies

8633 9577

2-amplicon: 8633 9112

fragment 1 480 bp 102 copies
(480 bp) 9577 102 copies
8945 9577
fragment 2 (633 bp)
 Rubella Sequencing Primers for 739 bp PCR
9112 Fragment
R
480b 9577R
p
8633 633bp
F
8945
F
• The 4 PCR primers can be used as sequencing primers, but
diluted to a different concentration than for PCR

• The sequences from each fragment are analyzed separately when

using Mega software and 2 consensus sequences are created

• The 2 sequences are combined to create 1 consensus sequence

to use for genotype analysis

• If using GeneStudio, the sequences are analyzed together

 Synthetic Positive Control RNA in CDC
Genotyping Kit
E1coding
region
Molecular Window (739-nt)
30-nt insertion

Pos control for

fragment 1

Pos control for

fragment 2

• 2 regions of the synthetic RNA contain deletions

• Fragments 1 and 2 positive control PCR products will
be about 80 nt smaller than wild-type RNA products
• The differences in product size between control and
wild-type allow for easy detection of contamination
1.5% Agarose Gels Showing Size Differences
in Control and Wild-type Products
Fragment 1 Fragment 2
1 2 M 1 2 M
1 2 M
1 - control RNA
2 - wild-type RNA
M- molecular marker

872bp
633bp
603bp
553bp
480bp
400bp
310bp
 Quality Control in Genotyping RT-PCR
(endpoint RT-PCR)
• Monitor assay controls. Always use positive and negative controls in all RT-
PCR assays.
• Use CDC positive controls (supplied in kits) to monitor for contamination from
positive controls.
• Maintain temperature control for storage of reagents and RNA.
• Maintain equipment, service regularly.
• Maintain directional workflow.
• The molecular weight marker should be clearly visible.
• The gel needs to run long enough to clearly determine the size of bands.
• Bands should be the expected size: approximately 630 bp for MeV amplicon;
approximately 480 bp for fragment 1 and 633 bp for fragment 2 RuV amplicons
• No extra bands (“primer dimers” are OK).
• Positive controls must show strong bands.
• If the negative control lane shows a band, the experiment must be repeated.
• Document all agarose gels with a photograph, including the marker.
• Can be e-mailed to regional reference laboratory or GSL for comments
 Sequence Quality Control (1)
Data quality cannot be determined by observation of the sequence text or
Mega files alone, must check the chromatogram.

>Menglian.CHN_09_d11
GTCAGTTCCACATTGGCATCTGAACTCGGTATCACTGCCGAGGATGCAAGGCTAGTTTCAGAGATTGCAATGCATACTACTGAGGCGGTTGGACCCAGACAAGCCCAA
GTGTCATTTCTACACGGTGATCAAAGTGAGAATGAGCTACCAGGATTGGGGGGCAAGGAAGACAGGAGGGCCAAACAGAGTCGGGGAGAATCCAGGGAGAATACCGGG
TCCAGCAGAGCAAGTGATGTGAGGGCTGCCCATCTTCCAACCAACACACCCCTAGACATTGACACCGCATCGGAGTCAGGCCAAGATCCGCAGGACAGTCGAAGGTCA
GCTGACGCCCTGCTCAGGCTGCAAGCCATGGCAGGAATCTTGGAAGAGCAAGGCTCAGACACGGACACCCCTAGGGTGTACAATGATAGAGATCTTCTAGAC

Example: two sequences from the same template. The right side shows high
quality sequence, the left side shows low quality. As a result of the low
quality, the bases are called differently.
 Sequence Quality Control (2)

• The chromatogram should have evenly spaced, sharp, well

defined peaks.

• Purification of sequencing products for the removal of salts and

excess fluorescent nucleotides. Extra fluorescent nucleotides
cause artefacts and ‘dye blobs’. Typically seen at 70bp and 120bp.
 Sequence Quality Control (3)

• Purification of PCR products for the removal of

excess primer and nucleotides is important for
unambiguous sequence determination.
• Presence of two primers in a sequencing
reaction leads to double peaks and unreadable
data.
 Sequence Quality Control (4)

• Low peaks throughout could indicate an

insufficient amount of DNA template.
• Inhibitory contaminant in samples (ethanol,
salts, etc)
• Insufficient amount of primer or too many freeze
thaws of primer stocks.
 Sequence Quality Control (5)

• Truncated sequence caused by too much template

• Truncated sequence caused by secondary structures in

GC rich regions
 Sequence Quality Control (6)

• Sudden large peaks covering 1-2 bases caused by

dried polymer in the capillary, degradation of polymer
or capillary
 Sequence Troubleshooting Solutions
• Be sure the PCR products are cleaned up to remove
unincorporated dNTPs, enzymes, and salts.
• Be sure the sequencing PCR is cleaned up properly to
remove excess salts, Big Dye, and ethanol.
• Add appropriate amounts of template to the reaction based
on either the gel photo or a concentration measurement.
• Keep track of the number of freeze/thaw cycles of the
primers.
• Ensure proper maintenance of the sequencing machine
occurs, including changing the buffer regularly, as well as
spatial and spectral calibrations.
• Once sequencing quality starts to degrade over time,
consider changing the polymer and/or capillary.
• pGEM sequencing control DNA template can help determine
whether failed reactions are caused by poor template quality
or sequencing reaction failure.
 Sequence Examples for Consideration

How many Ts? Is it two or three? What

should be done to determine the correct call?
Which of the sequences is correct? Is there
one A or two?
All three of the sequences are good quality,
which one do we believe and why?
How do we feel about believing the nucleotide
“below” in some instances but not in others?
What should be done?
Our sequencing window is only 449
nucleotides, where is the other base?
Our sequencing window is 451 nucleotides,
where is the extra base?
 AB Sequencing and GeneStudio Analysis Programs
both display chromatograms or sequences with quality values

Quality values (QVs) ≥20 are colored blue

and considered acceptable. QVs of 15-19 are
yellow, QVs of 1-14 are red. QVs of less than
20 are not acceptable.
The height of the bar indicates how high the
QV is.

If the quality values for a base are low in all sequences,

repeat the sequencing reactions.
 Sequencing Analysis Programs

• Review the quality values for the entire sequence.

• The higher the bar, the better the quality.
• Blue bars have acceptable quality.
• The beginning and end of a sequence may not be required
for analysis if these nucleotides are outside of the
sequencing window.
 Cropping the Sequence: Measles
The genotyping window is smaller than the PCR fragment
MeV216 (nt 1105-1124)
1105 TGG AGC TAT GCC ATG GGA GTA GGA GTG GAA CTT GAA AAC TCC ATG GGA 1152
1153 GGT TTG AAC TTT GGC CGA TCT TAC TTT GAT CCA GCA TAT TTT AGA TTA 1200
 start of N-450 window
1201 GGG CAA GAG ATG GTA AGG AGG TCA GCT GGA AAG GTC AGT TCC ACA TTA 1248
1249 GCA TCT GAA CTC GGT ATC ACT GCC GAG GAT GCA AGG CTT GTT TCA GAG 1296
1297 ATT GCA ATG CAT ACT ACT GAG GAC AAG ATC AGT AGA GCG GTT GGA CCC 1344
1344 AGA CAA GCC CAA GTA TCA TTT CTA CAC GGT GAT CAA AGT GAG AAT GAG 1392
1394 CTA CCG AGA TTG GGG GGC AAG GAA GAT AGG AGG GTC AAA CAG AGT CGA 1440
1441 GGA GAA GCC AGG GAG AGC TAC AGA GAA ACC GGG CCC AGC AGA GCA AGT 1488
1489 GAT GCG AGA GCT GCC CAT CTT CCA ACC GGC ACA CCC CTA GAC ATT GAC 1536
1537 ACT GCA TCG GAG TCC AGC CAA GAT CCG CAG GAC AGT CGA AGG TCA GCT 1584
1585 GAG CCC CTG CTT AGG CTG CAA GCC ATG GCA GGA ATC TCG GAA GAA CAA 1632
1633 GGC TCA GAC ACG GAC ACC CCT ATA GTG TAC AAT GAC AGA AAT CTT CTA 1680
 end of N-450 window MeV214
1681 GAC TAG GTG CGA GAG GCC GAG GGC CAG AAC AAC ATC CGC CTA CCC TCC 1728
1729 ATC ATT GTT ATA AAA AA
 Measles Phylogeny
MVi/New York.USA/0.94 (B3)
MVi/Ibadan.NGE/0.97/1 (B3)
450nt MVi/Yaounde.CMR/12.83 (B1)
MVi/Libreville.GAB/0.84 (B2)
sequence MVi/Maryland.USA/0.54 (A)
window of MVs/Madrid.ESP/0.94 (F)(SSPE)
MVi/Goettingen.DEU/0.71 (E)
the 28 WHO MVi/Tokyo.JPN/0.84 (C1)
MVi/Maryland.USA/0.77 (C2)
reference MVi/Erlangen.DEU0.90 (C2)
MVi/Hunan.CHN/0.93/7 (H1)
viruses MVi/Beijing.CHN/0.94/1 (H2)
MVi/Berkeley.USA/0.83 (G1)
MVi/Amsterdam.NLD/49.97 (G2)
MVi/Gresid.IDN/17.02 (G3)
MVi/Bristol.GBR/0.74 (D1)
MVi/New Jersey.USA/0.94/1 (D6)
MVi/Johannesburg.ZAF/0.88/1 (D2)
MVi/Kampala.UGA/51.01/1 (D10)
MVi/Victoria.AUS/16.85 (D7)
MVi/Illinois.USA/50.99 (D7)
MVi/Menglian.Yunnan.CHN/47.09 (D11)
MVi/Manchester.GBR/30.94 (D8)
MVi/Montreal.CAN0.89 (D4)
MVi/Victoria.AUS/12.99 (D9)
MVi/Illinois.USA/0.89/1 (D3)
MVi/Bangkok.THA/0.93/1 (D5)
MVi/Palau.PLW/0.93 (D5)

5
 Cropping the Sequence: Rubella
The genotyping window is smaller than the PCR fragment
8633F
8581 CTACAAGCAGTACCACCCTACCGCGTGCGAGGTTGAACCTGCCTTCGGACACAGCGACGC 8640

8641 GGCCTGCTGGGGCTTCCCCACCGACACCGTGATGAGCGTGTTCGCCCTTGCTAGCTACGT 8700

739 start
8701 CCAGCACCCTCACAAGACCGTCCGGGTCAAGTTCCATACAGAGACCAGGACCGTCTGGCA 8760

8761 ACTCTCCGTTGCCGGCGTGTCGTGCAACGTCACCACTGAACACCCGTTCTGCAACACGCC 8820

8821 GCACGGACAACTCGAGGTCCAGGTCCCGCCCGACCCCGGGGACCTGGTTGAGTACATTAT 8880

8881 GAATTACACCGGCAATCAGCAGTCCCGGTGGGGCCTCGGGAGCCCGAATTGCCACGGCCC 8940

8945F
8941 CGATTGGGCCTCCCCGGTTTGCCAACGCCATTCCCCTGACTGCTCGCGGCTTGTGGGGGC 9000

9001 CACGCCAGAGCGCCCCCGGCTGCGCCTGGTCGACGCCGACGACCCCCTGCTGCGCACTGC 9060

9112R
9061 CCCTGGACCCGGCGAGGTGTGGGTCACGCCTGTCATAGGCTCTCAGGCGCGCAAGTGCGG 9120

9121 ACTCCACATACGCGCTGGACCGTACGGCCATGCTACCGTCGAAATGCCCGAGTGGATCCA 9180

9181 CGCCCACACCACCAGCGACCCCTGGCATCCACCGGGCCCCTTGGGGCTGAAGTTCAAGAC 9240

9241 AGTTCGCCCGGTGGCCCTGCCACGCACGTTAGCGCCACCCCGCAATGTGCGTGTGACCGG 9300

9301 GTGCTACCAGTGCGGTACCCCCGCGCTGGTGGAAGGCCTTGCCCCCGGGGGAGGCAATTG 9360

9361 CCATCTCACCGTCAATGGCGAGGACCTCGGCGCCGTCCCCCCTGGGAAGTTCGTCACCGC 9420

739 end
9421 CGCCCTCCTCAACACCCCCCCGCCCTACCAAGTCAGCTGCGGGGGCGAGAGCGATCGCGC 9480

9481 GACCGCGCGGGTCATCGACCCCGCCGCGCAATCGTTTACCGGCGTGGTGTATGGCACACA 9540

9577R
9541 CACCACTGCTGTGTCGGAGACCCGGCAGACCTGGGCGGAGTGGGCTGCTGCCCATTGGTG 9600
 Rubella RV i Dezhou.CHN 02 [1 E] 1E

739nt sequence
RV i MYS 01 [1E]
Reference virus sequences for
the updated rubella nomenclature RV i Tokyo .JPN 90 [1D]
1D

window of
RV i S aita ma.JPN 94 [1 D]

Sequences consist of 739 RV i Tochigi.JPN 0 4 [1j]

nucleotides in the E1 coding 1J1j
the 32 WHO
region RV i DalyCity.CA.USA 97 [1j]

RV i Li nqing.CHN 00 [1 F]
1F

reference
RV i Dang sha n.CHN 00 [1F]

RV i P A.USA 64 [1a] VAC

viruses
RV i Toyama.JP N 67 [1a]

RV i Tiber ias.ISR 8 8 [1B]

RV i Jerusale m.ISR 7 5 [1B] 1B

RV i B eneBer ak.ISR 7 9 [1B]

RV i P avia .ITA 2 1.9 1 [1i]

Clade 1
1I
RV i Milan.ITA 4 6.9 2 [1i]

RV i Minsk.BL R 28.05 [1h]

1h
1H
RV i Tomsk.RUS 04 [1h ]

RV i UGA 20 .01 [1G]

RV i O nta rio.CA N 05 [1G] 1G

RV i Minsk.BL R 29.04 [1G]

RV i P AN 99 [1C]

RV i S LV 02 [1C] 1C

8-10% RV i LA .USA 91 [1C]

RV i NJ.USA 61 [1a] V AC

difference RV i B EL 63 [1a ] VAC

RV i Moscow.RUS 67 [2C]

between RV i Moscow.RUS 97 [2C]

2C

RV i B eijing.CHN 80 [2 A] VA C
Clade 2
Clades 1 RV i B eijing.CHN 79 [2 A]

RV i TelA viv.ISR 68 [2B ]

2A

and 2 RV i A nqing.CHN 00 [2 B]

RV i S eattle.WA .USA 16.00 [2B] 2B

 Measles phylogenetic tree made in Mega
MVs/California.USA/50.14 SSPE
MVi/Montreal.CAN0.89 (D4)
MVi/New York.USA/0.94 (B3) MVi/Victoria.AUS/12.99 (D9)
MVi/Ibadan.NGE/0.97/1 (B3) MVi/Illinois.USA/0.89/1 (D3)
MVi/Yaounde.CMR/12.83 (B1)
MVi/Bangkok.THA/0.93/1 (D5)
MVi/Libreville.GAB/0.84 (B2)
MVi/Palau.PLW/0.93 (D5)
MVi/Maryland.USA/0.54 (A)
MVs/Madrid.ESP/0.94 (F)(SSPE) MVi/Manchester.GBR/30.94 (D8)
MVi/Goettingen.DEU/0.71 (E) MVi/Menglian.Yunnan.CHN/47.09 (D11)
MVi/Tokyo.JPN/0.84 (C1) MVi/Victoria.AUS/16.85 (D7)
MVi/Maryland.USA/0.77 (C2) MVi/Illinois.USA/50.99 (D7)
MVi/Erlangen.DEU0.90 (C2)
MVi/Johannesburg.ZAF/0.88/1 (D2)
MVi/Berkeley.USA/0.83 (G1)
MVi/Kampala.UGA/51.01/1 (D10)
MVi/Amsterdam.NLD/49.97 (G2)
MVi/Gresid.IDN/17.02 (G3) MVi/Bristol.GBR/0.74 (D1)
MVi/Bristol.GBR/0.74 (D1) MVi/New Jersey.USA/0.94/1 (D6)
MVi/New Jersey.USA/0.94/1 (D6) MVi/Maryland.USA/0.54 (A)
MVs/California.USA/50.14 SSPE MVi/Maryland.USA/0.77 (C2)
MVi/Montreal.CAN0.89 (D4) MVi/Erlangen.DEU0.90 (C2)
MVi/Victoria.AUS/12.99 (D9)
MVi/Tokyo.JPN/0.84 (C1)
MVi/Bangkok.THA/0.93/1 (D5)
MVs/Madrid.ESP/0.94 (F)(SSPE)
MVi/Palau.PLW/0.93 (D5)
MVi/Illinois.USA/0.89/1 (D3) MVi/Goettingen.DEU/0.71 (E)
MVi/Victoria.AUS/16.85 (D7) MVi/Libreville.GAB/0.84 (B2)
MVi/Illinois.USA/50.99 (D7) MVi/Yaounde.CMR/12.83 (B1)
MVi/Manchester.GBR/30.94 (D8) MVi/New York.USA/0.94 (B3)
MVi/Menglian.Yunnan.CHN/47.09 (D11)
MVi/Ibadan.NGE/0.97/1 (B3)
MVi/Johannesburg.ZAF/0.88/1 (D2)
MVi/Berkeley.USA/0.83 (G1)
MVi/Kampala.UGA/51.01/1 (D10)
MVi/Hunan.CHN/0.93/7 (H1) MVi/Amsterdam.NLD/49.97 (G2)
MVi/Beijing.CHN/0.94/1 (H2) MVi/Gresid.IDN/17.02 (G3)
MVi/Hunan.CHN/0.93/7 (H1)
5 MVi/Beijing.CHN/0.94/1 (H2)
Make
sure to
unclick
the
topology
button!
 Phylogenetic Analysis using Mega

Considerations:
 Reference strains: do you have the complete WHO
reference strains in a .fas file format ready to
import?
 Named strains: do you have the named strains of
the genotype of interest in a .fas format, ready to
import?
 Other strains of interest: is this part of an ongoing
outbreak or are there strains from a neighboring
country that you would like to compare to your
currently circulating strains?
 File Types for Genotype Analysis
>Menglian.CHN_09_d11
GTCAGTTCCACATTGGCATCTGAACTCGGTATCACTGCCGAGGATGCAAG
GCTAGTTTCAGAGATTGCAATGCATACTACTGAGGACAGGACCAGTAGAG
CGGTTGGACCCAGACAAGCCCAAGTGTCATTTCTACACGGTGATCAAAGT
GAGAATGAGCTACCAGGATTGGGGGGCAAGGAAGACAGGAGGGCCAAACA
GAGTCGGGGAGAATCCAGGGAGAGCTACAGAGATACCGGGTCCAGCAGAG Chromatogram file can only be
CAAGTGATGTGAGGGCTGCCCATCTTCCAACCAACACACCCCTAGACATT
GACACCGCATCGGAGTCAGGCCAAGATCCGCAGGACAGTCGAAGGTCAGC
opened by specialized sequence
TGACGCCCTGCTCAGGCTGCAAGCCATGGCAGGAATCTTGGAAGAGCAAG analysis software
GCTCAGACACGGACACCCCTAGGGTGTACAATGATAGAGATCTTCTAGAC • Mega can read ABI files (.ab1)
>MVP.UK_74_D1
GTCAGCTCCACATTGGCATCTGGACTCGGTATCACTGCCGAGGACGCAAG • Mega files (.meg)
GCTTGTTTCAGAGATTGCAATGCATACTACTGAGGACAGGATCAGTAGAG • FASTA format (.fas)
CGGTTGGACCCAGACAAGCCCAAGTGTCATTTCTACACGGTGATCAAAGT
GAGAATGAGCTACCAGGATTGGGGGGCAAGGAAGACAGGAGGGTCAAACA • Text files (.txt)
GAGTCGAGGAGAAGCCAGGGAGAGCTACAGAGATACCGGGTCCAGCAGAG
CAAGTGATGCAAGAGCTGCCCATCTTCCAACCAGCACACCCCTAGACATT
GACACTGCATCGGAGTCAAGCCAAGATCCTCAGGACAGTCGAAGGTCAGC
TGACGCCCTGCTCAGGCTGCAAGCCATGGCAGGAATCTCGGAAGAACAAG Examples show cropped
GCTCAGACACGGACACCCCTCGAGTGTACAATGACAGAGATCTTCTAGAC
>BRAXATOR.DEU_71_E sequences of 450 nt
GTCAGTTCCACATTGGCATCTGAACTCGGTATCACTGCCGAGGATGCAAG
GCTTGTTTCGGAGATTGCAATGCACACTACTGAGGACAGGATCAGCAGAG
CGGTTGGACCCAGACAAGCCCAAGTGTCATTTCTACACGGTGATCAAAGT
GAGAATGAGCTGCCGAGATGGGGGGGCAAGGAAGATAGGAGGGCCAAACA
GAGTCGAGGAGAAGCCAGGGAGATCTACAGAGAAACCGGGCCCAGCAGAG
CAAGTGATGCGAGAGCTGCCCATCTTCCAACCGGCACACCCCTAGACATT
GACACTGCATCGGAGTCCAGCCAAGATCAGCAGGACAGTCAAAGGTCAGC
TGACGCCCTACTCAGGCTGCAAGCCATGGCAGGAATCTCGGAAGAACAAG
GCTCAGACACGGACACCCCTAGAGTGTACAATGACAGAGATCTTCTAGAC
 Acknowledgements

Thank you for your attention!

National Center for Immunization & Respiratory Diseases

Measles, Mumps, Rubella & Herpes Virus Laboratory Branch