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Sample tracking using
plasmid barcodes
Cristian Pérez García
Bioinformatician

2© 2016 IMEGEN – Información confidencial. Todos los derechos reservados.
We regularly do clinical diagnosis

ENAC Acreditation
Asked for:
- The ENAC acreditation of
NextGeneDX (amplicon)
analysis.
- The ENAC acreditation of
clinical exome analysis

But we had an Issue: sample
traceability
• We until that moment did not traced back our samples from the
VCF file to the DNA used for NGS.
• ENAC wisely asked to have biological sample tracking enabled
in our lab (LIMS is not enough).
Resulting
Genotype
Blood
stock
DNA
dilution
SequencingWet lab

But we had an issue: sample
traceability
• We until that moment did not traced back our samples from the
VCF file to the DNA used for NGS.
• ENAC wisely asked to have biological sample tracking enabled
in our lab (LIMS is not enough).
Resulting
Genotype
Blood
stock
DNA
dilution
SequencingWet lab

Reasons to have a sample
tracking system
Consequent to sample mix-ups in a research setting, erroneous data and
sample matching may result in:
• Case-control: a loss of power for identification of causal variants.
• Clinical-context: This may lead to delayed or inaccurate reporting of
results to patients.
Whilst good practice in the handling of samples and increased laboratory
automation minimizes potential for error, additional checkpoints are still
required to support quality control.
A method for post hoc confirmation of sample identity is therefore
highly desirable.

Which options do we have?
• SNPs in Introns
• Sequenom: ±24 SNPs in
“pangenomic regions”.
• SNPs in Exons Capture
• KASP: exonic SNPs in regions used
in capture methods
• Ampliseq
• STRs or indels

Are any of these suitable
for us?
Which options do we have?

Choose SNPs in the region of
the capture probes.
• SNPs in Introns
• Sequenom: ±52 SNPs, some
in introns.
• KASP: exonic SNPs in regions
used in capture methods
• Ampliseq

Choose SNPs in the region of
the capture probes.
• SNPs in Introns
• Sequenom: ±52 SNPs, some
in introns
• KASP: exonic SNPs in regions
used in capture methods
• Ampliseq

STRs or Indels
We don’t have a Sequenom, why don’t use STRs (Single
Tandem Repeats) or indels and check with fragment length
analysis?

- Expensive
Disadvantages

- Expensive
- Time Consuming
Disadvantages

- Expensive
- Time Consuming
- Extra steps for our pipeline
(genotyping)
Disadvantages

• These are for tracking sample identity across
multiple experiments.
Disadvantages

• These are for tracking Sample identity across
multiple experiments.
• This methods are not suitable for amplicon
sequencing, without needing to do another
genotyping step.
Disadvantages

Two weeks of thinking
and lots of coffee
breaks brain storming
But, is there a cheaper way to
do this…?

• Why not adding a sequence that is captured by the TSO?
• Nice idea, but....
• Should we need to use other approach with amplicons?
But, is there a cheaper way to
do this…?

Let’s think again....
We have two workflows:
• Exoma capture
• Amplicons (Nextera after PCR)

Sample Tracking with Plasmids
• We can build a plasmid whose
insert is a region of a gene
with a probe in TruSight One
and add some barcodes in the
middle of the sequence.
• PCR primers in both ends of
the sequence.

Sample Tracking with Plasmids
• We can build a plasmid whose
insert is a region of a gene
with a probe in TruSight One
and add some barcodes in the
middle of the sequence.
• PCR primers in both ends of
the sequence.
This method should work
for both workflows!

Selecting the region of interest
Find a captured region in TSO of not
clinical interest with good capture

• Which one to use?
• We are not reporting 3’-UTR miRNA
target sites still has unclear diagnostic
significance except for very specific
cases.
Selecting the region of interest
TMEM-135 3’UTR (800x)

Building the plasmid
• All tubes of NextGenDX amplicons
would have a sequence with NGS
adapters so all tubes would report
this barcode
• The barcode would be also captured
and sequenced by TSO
STID:0204

Building the plasmid
CP-N701
acgtaccgtagaactagcgactgcCATTGGTGCCAGCTCTAT
AATTTCTTCTTCTTGTGGAATTAACAAAGAAAGGAG
TGTCAAGGACTGAGATGACCCTCAGATTGGGGGG
CTGTCTTAGATTCTAGGGCTTTGTAGTACTATGTTT
CTGTTTAAAGTAGTGGCCTCAGGTGACTTTGTAAT
AGCCCTGTAGTTGCAAAAAGGTCGCCTTAGTAACT
ACAAAGAAATGAAACTGACTCTAGTGTGTGTGACT
TCTGGAAACAGAAGTGGGGCAGTAAGTTGGCCAT
GATATAGCTAGTGTCATAGGACTACAGCAGAGTAG
TGAGTGAATGGCCTTAAGCTTACAGCTGTGGTGAA
TAAGAATGTGTGCTATTTTACACACAGAAGAATggat
cttcgattccatctgactgt

Testing concentrations
Exome Amplicon

Mapping and testing
[cpg@dream3 plasmidos]$ python3.4 checkPlasmids.py --
folder 151209_NS500802_0062_AH5KF3AFXX-
NxSqEx74/23732
Mapping...
# Searching for reads aligned with the plasmids references
CP-N701 0
CP-N702 1
CP-N703 179
CP-N704 115
CP-N705 0
CP-N706 0
CP-N707 0
CP-N710 0

Results

Amplicon test

Exome test

Expected results

No plasmid added into sample

Sample Swap

35
Conclusions
• We have build a new, cheap and relatively simple
technique for sample tracking.
• This technique does not require extra lab steps so that
the protocol won’t take any longer.
• The sample tracking information is in the output data, so
when evaluating the results, we can evaluate if sample
swap has occurred.

Instituto de Medicina Genómica SL
Agustín Escardino 9,
Parc Científic de la Universitat de València
46980 Paterna (Valencia, España)
+34 963 212 340
Imegen.es
© 2015 IMEGEN – Información confidencial. Todos los derechos reservados.

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