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Table 1 Common pre-analytical and sample preparation issues related to different sample types

From: Technological considerations for genome-guided diagnosis and management of cancer

Sample type Common issues Impact Contingencies/solutions
Formalin-fixed, paraffin-embedded (FFPE) • Low yield of DNA
• DNA degradation
• DNA base modification
• RNA degradation
• Reduced complexity libraries; library failure; decreased sensitivity
• Reduced complexity; library failure; decreased sensitivity
• Increased false positive rate
• Library failure; high duplication
• DNA repair; pooling of indexed libraries prior to capture (exomes or panels); specialized low input library methods
• DNA repair; short amplicon amplification; specialized library methods
• FFPE-aware filtering of variants; DNA repair
• Selection-based or targeted preparation instead of polyA-based preparation
Fresh frozen tissue of bulk cells • Buffer or process-induced modification of DNA bases • Increased false positive rate • Chelation of oxidative species; oxidation aware filtering
Single cells • Low DNA yield
• Whole genome amplification (WGA) bias
• Low RNA yield
• Library failure
• Increased false positives and false negatives
• Library failure
• Optimized WGA
• Whole transcriptome amplification (WTA)
Liquid biopsy • Low DNA yield of cfDNA
• Low purity of ctDNA in cfDNA
• Low DNA yield from CTCs
• Low RNA yield and quality from CTCs
• Low RNA yield from EVs
• Library failure; reduced sensitivity
• Reduced sensitivity
• Library failure; reduced sensitivity; reduced specificity
• Library failure
• Library failure
• Optimized library preparation; specialized library preparation
• High sequencing depth; molecular barcoding (UMIs)
• WTA; specialized library preparation.
• WTA; specialized library preparation.