br Results br NGS of CRC
NGS of CRC Samples
The choice of sample types to be analyzed by NGS is dictated by availability or clinical questions. For instance, most often formalin-fixed paraffin-embedded (FFPE)-derived DNA samples are available for retrospective studies, whereas tumor heterogeneity, or longitu-dinal monitoring and detection of minimal residual disease, is often assessed using plasma ctDNA.23,44 Sometimes, fresh tissues (such as biopsies and preclinical models) can also be available (Figure 1A). It is therefore of utmost relevance to be able to process a variety of different samples. To this aim, we outline below specific guidelines for the initial steps of sample preparation.
First of all, we identified nucleic ATPγS tetralithium salt isolation as a crucial step that requires tailored protocols and specific kits for each sample type (Figure 1C) to generate DNA of suitable quality and quantity for further analyses (Figure 1B).
After DNA extraction, samples authentication with short tandem repeat analysis is performed to avoid misidentification and to verify the correct matching among samples belonging to the same patient/ preclinical model.
Because DNA displays distinct characteristics depending on the starting material (Figure 1B), we adapted and optimized sample processing and sequencing protocols according to sample types.
In our experience, FFPE-derived DNA typically shows poor quality, likely associated with the processing steps of histology specimen preparation. In addition, the presence of DNA fragments of variable length makes this type of sample the most challenging to
process with the NGS workflow. We obtained a remarkable improvement in the quality of final sequencing results by me-chanical shearing of DNA, thus rendering fragment length homo-geneous and tailored for Illumina sequencers. The next steps involve end-repair and A-tailing of fragmented DNA molecules, both essential for subsequent ligation of adapter sequences (Figure 1D).
Unlike FFPE-derived DNA, ctDNA displays good quality owing to the absence of chemical contaminants, but it is highly frag-mented. In light of this, in our protocol, ctDNA is directly sub-jected to end-repair and A-tailing steps prior to adapter ligation (Figure 1D).
Finally, intact high-quality DNA is usually isolated from fresh or frozen tissue. We enzymatically fragment this type of DNA by means of a transposon that cuts and simultaneously inserts sequencing adapters (Figure 1D).
In all cases, index sequences specific for each sample are inserted by means of a short PCR amplification, thus allowing to pool several samples in the same library.
The approaches outlined above allow isolation of DNA fragments of suitable length with ligated adapters. At this point, all samples could be directly subjected to whole genome sequencing (WGS). However, as compared with WGS, the analysis of specific regions of interest, such as WES or custom panels, provides several advantages, as discussed later. We found that the capture-based approach is the preferred choice for enrichment of target regions, because it in-troduces less intrinsic biases compared with amplicon-based strate-gies.45 In capture-based approaches, specifically designed biotinylated probes that hybridize to the corresponding target se-quences are then captured by streptavidin magnetic beads (Figure 1E). The enriched libraries are then subjected to a final short PCR amplification and, afterwards, loaded on the sequencer.
Genotyping CRCs in Blood
We and others have shown that liquid biopsies can complement and, in some instances, provide more information than standard tissue biopsies in patients with advanced CRC.35 Analyses of plasma samples offer the possibility to obtain a broad range of information on tumor heterogeneity and clonal molecular dynamics from a blood withdrawal. Notably, the workload required for plasma pro-cessing takes significantly less time than preparation of FFPE sam-ples (Figure 2A). Importantly, to preserve ctDNA in plasma, blood samples must be processed within 2 to 4 hours from collection. After blood centrifugation and plasma isolation, ctDNA can be extracted within a few hours. Overall, ctDNA can be available for downstream analyses within 24 to 36 hours from sample collection. This aspect is important as, in some instances, the timeline required to generate molecular maps starting from ctDNA or genomic DNA extracted from FFPE tissue has clinical relevance.