Dataset Information

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 SureSelectQXT 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 SureSelectQXT libraries. Sequencing results showed that, using our modified SureSelectQXT 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.

SUBMITTER: Rosa-Rosa JM

PROVIDER: S-EPMC6182866 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Modified SureSelect<sup>QXT</sup> Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples.

Rosa-Rosa J M JM Caniego-Casas T T Leskela S S Muñoz G G Del Castillo F F Garrido P P Palacios J J

Biological procedures online 20181012

<h4>Background</h4>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.).<h4>Results</h4>We performed several experiments to underst ...[more]

PMID: 30337841

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Project description:Objective: RNA extraction and library preparation from formalin-fixed, paraffin-embedded (FFPE) samples are crucial pre-analytical steps towards achieving optimal downstream RNA sequencing (RNASeq) results. In this study, we assessed 2 Illumina library preparation methods for RNA-Seq analysis using archived FFPE samples from human cancer indications at 2 independent vendors. Methods: Twenty-five FFPE samples from 5 indications (non-small cell lung cancer, colorectal cancer, renal carcinoma, breast cancer, and hepatocellular carcinoma) were included, covering a wide range of sample storage durations (3-25 years-old), sample qualities, and specimen types (resection vs core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20 pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry in paired-end mode with a read length of 2 × 50 bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions for each gene in each sample to compare 2 library kits at 2 different vendors. Results: Our data showed that TruSeq RNA Access libraries yield over 80% exonic reads across different quality samples, indicating higher selectivity of the exome pull down by the capture approach compared to the random priming of the TruSeq Stranded Total kit. The overall QC data for FFPE RNA extraction, library preparation, and sequencing generated by the 2 vendors are comparable, and downstream gene expression quantification results show high concordance as well. With the TruSeq Stranded Total kit, the mean Spearman correlation between vendors was 0.87 and the mean Pearson correlation was 0.76. With the TruSeq RNA Access kit, the mean Spearman correlation between vendors was 0.89 and the mean Pearson correlation was 0.73. Interestingly, examination of the cross-vendor correlations compared to various common QC statistics suggested that library concentration is better correlated with consistency between vendors than is the RNA quantity. Conclusions: Our analyses provide evidence to guide selection of sequencing methods for FFPE samples in which the sample quality may be severely compromised.

Dataset Information

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

Publications

Modified SureSelect<sup>QXT</sup> Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples.

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