Table 1.
DNA Quality Guidelines by Analytical Platform
| Application | Criteria employed | Reference |
|---|---|---|
| NGS | Monitor yield after library preparation and mean insert size as predictors for characterization success. Age of sample is not a predictor of success, as fixation technique plays a greater role. | BROAD Institute (http://genomics.broadinstitute.org/data-sheets/DTS_FFPE_4-2017.pdf) |
| A minimum of 100 ng DNA is used for library construction. DNA integrity is not assessed by gel electrophoresis. Library metrics are used to determine pass/fail status before sequencing. Successful libraries should have the majority of library fragments between 300 and 600 bp in size with a minimum yield of 15 μL at 3 nM. | Dr. Andrew Mungall BC Cancer (www.bcgsc.ca/services/sequencing-libraries-faq) | |
| Sample intake QC-minimum DNA integrity (>200 bp) and absence of protein contamination evaluated by E-Gel. | Dr. Harsha Doddapaneni and Dr. David Wheeler (Baylor College of Medicine Human Genome Sequencing Center) | |
| Library construction yields should be >300 ng with fragments between 200 and 800 bp when using 100 ng input (manual preparation) or 250 ng (robotic preparation). | ||
| Post-library capture should have >10 nM yield and devoid of primer dimers. | ||
| 150 ng double-stranded DNA, amplification of 100 bp product; ΔCt <2 using the FFPE QC Kit | Personal communication Dr. Betsou (IBBL, Luxembourg) | |
| >6% amplifiable copies; Input adjusted based on PCR amplification of TBP or FTH1 | Sah et al.22 | |
| ΔCt <1.55 real-time PCR-based Illumina FFPE QC Kit | Serizawa et al.23 | |
| PCR | Comparative assessment of differentially sized GAPDH PCR amplicons: 100, 236, 299, 411, bp visualized by HPLC | Wang et al.24 |
| aCGH | Amplification of a 200 bp fragment of GAPDH from 100 ng DNA | van Beers et al.25 |
| Amplification of 200 bp product; >2 μg DNA | Personal Communication Dr. Betsou (IBBL, Luxembourg) |
aCGH, array comparative genomic hybridization; FFPE, formalin-fixed paraffin-embedded; FTH1, ferritin heavy polypeptide 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HPLC, high-performance liquid chromatography; NGS, next-generation sequencing; PCR, polymerase chain reaction; TBP, tata-binding protein.