Modified SureSelectQXT Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples

Abstract

Background: Personalised medicine is nowadays a major objective in oncology. Molecular characterization of tumours through NGS offers the possibility to find possible therapeutic targets in a time- and cost-effective way. However, the low quality and complexity of FFPE DNA samples bring a series of disadvantages for massive parallel sequencing techniques compared to high-quality DNA samples (from blood cells, cell cultures, etc.).

Results: We performed several experiments to understand the behaviour of FFPE DNA samples during the construction of SureSelect^QXT libraries. First, we designed a quality checkpoint for FFPE DNA samples based on the quantification of their amplification capability (qcPCR). We observed that FFPE DNA samples can be classified according to DIN value and qcPCR concentration into unusable, or low-quality (LQ) and good-quality (GQ) DNA. For GQ samples, we increased the amount of input DNA to 150 ng and the digestion time to 30 min, whereas for LQ samples, we used 50 ng of DNA as input but we decreased the digestion time to 1 min. In all cases, we increased the cycles of the pre-hyb PCR to 10 but decreased the cycles of the post-hyb PCR to 8. In addition, we confirmed that using half of the volume of reagents can be beneficial. Finally, in order to obtain better results, we designed a decision flow-chart to achieve a seeding concentration of 12-14 pM for MiSeq Reagent Kit v2.

Conclusions: Our experiments allowed us to unveil the behaviour of low-quality FFPE DNA samples during the construction of SureSelect^QXT libraries. Sequencing results showed that, using our modified SureSelect^QXT protocol, the final percentage of usable reads for low-quality samples was increased more than three times allowing to reach median depth/million reads values of 76.35. This value is equivalent to ~ 0.9 and ~ 0.7 of the values obtained for good-quality FFPE and high-quality DNA respectively.

Keywords: FFPE samples; NGS; Optimization; Protocol; SureSelectQXT.

Fig. 1

Quality and integrity of the 9 DNA samples with the Agilent 2200 Tape Station and Genomic DNA Screen Tape. a Genomic DNA samples. b Digested, amplified and purified DNA samples

Fig. 2

Quantification and qualification of library DNA with the Agilent 2200 Tape station and D1000 Screen Tape using 50 (1), 150 (2) and 500 (3) ng of DNA input. a Gel image. b Electropherogram. c Table with library concentration estimations

Fig. 3

Quantification and qualification of library DNA with the Agilent 2200 Tape station and D1000 Screen Tape using 8 (1) or 10 (2) cycles during pre-hybridization PCR. a Gel image. b Electropherogram. c Table with library concentration estimations

Fig. 4

Gel image of NA12892, NT1 and T1 pre-hyb products with the Agilent 2200 Tape station and D1000 Screen Tape. Input DNA was 50 ng. a Using standard reagent volumes. b Using ½x standard reagent volumes

Fig. 5

Pre-hyb library quantification using the Agilent 2200 Tape station and D1000 Screen Tape. a Gel image showing library products where digestion times were set up at 1, 2, 3, 4, 5 and the standard 10 min. b Graphical representation of pre-hyb library concentration vs. digestion times. c Gel image of different DNA input and digestion time tests. b DNA input and digestion time conditions and pre-hyb concentration

Fig. 6

Correlations. a Correlation between post-hyb concentration measured by QPCR and DIN value for all samples. b Correlation between post-hyb concentration measured by QPCR and DIN value for FFPE samples. c Correlation between post-hyb concentration measured by QPCR and qcPCR concentration measured with Tape Station for FFPE samples. d Correlation between post-hyb concentration and qcPCR concentration measured, both with Tape Station, for FFPE samples