presentacion jbi

Sample tracking using plasmid barcodes

Cristian Pérez GarcíaBioinformatician

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 theVCF file to the DNA used for NGS.

• ENAC wisely asked to have biological sample tracking enabledin 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 theVCF file to the DNA used for NGS.

• ENAC wisely asked to have biological sample tracking enabledin 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.

• SNPs in Exons Capture• 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

• SNPs in Exons Capture• KASP: exonic SNPs in regions

used in capture methods

• Ampliseq


STRs or Indels

We don’t have a Sequenom, why don’t use STRs (SingleTandem Repeats) or indels and check with fragment lengthanalysis?


- 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 ampliconsequencing, without needing to do another genotyping step.

Disadvantages


Two weeks of thinkingand 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 whoseinsert is a region of a genewith a probe in TruSight Oneand add some barcodes in themiddle of the sequence.

• PCR primers in both ends ofthe sequence.


Sample Tracking with Plasmids

• We can build a plasmid whoseinsert is a region of a genewith a probe in TruSight Oneand add some barcodes in themiddle of the sequence.

• PCR primers in both ends ofthe 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 miRNAtarget 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 ampliconswould have a sequence with NGSadapters so all tubes would reportthis barcode

• The barcode would be also captured and sequenced by TSO

STID:0204


Building the plasmid

CP-N701

acgtaccgtagaactagcgactgcCATTGGTGCCAGCTCTATAATTTCTTCTTCTTGTGGAATTAACAAAGAAAGGAGTGTCAAGGACTGAGATGACCCTCAGATTGGGGGGCTGTCTTAGATTCTAGGGCTTTGTAGTACTATGTTTCTGTTTAAAGTAGTGGCCTCAGGTGACTTTGTAATAGCCCTGTAGTTGCAAAAAGGTCGCCTTAGTAACTACAAAGAAATGAAACTGACTCTAGTGTGTGTGACTTCTGGAAACAGAAGTGGGGCAGTAAGTTGGCCATGATATAGCTAGTGTCATAGGACTACAGCAGAGTAGTGAGTGAATGGCCTTAAGCTTACAGCTGTGGTGAATAAGAATGTGTGCTATTTTACACACAGAAGAATggatcttcgattccatctgactgt


Testing concentrations

Exome Amplicon


Mapping and testing

[cpg@dream3 plasmidos]$ python3.4 checkPlasmids.py --folder 151209_NS500802_0062_AH5KF3AFXX-NxSqEx74/23732Mapping...# Searching for reads aligned with the plasmids referencesCP-N701 0CP-N702 1CP-N703 179CP-N704 115CP-N705 0CP-N706 0CP-N707 0CP-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 SLAgustín Escardino 9, Parc Científic de la Universitat de València46980 Paterna (Valencia, España)+34 963 212 340

Imegen.es

© 2015 IMEGEN – Información confidencial. Todos los derechos reservados.

presentacion jbi

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