A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single-Nucleotide Variant Detection.
Author(s): Lee C, Bae JS, Ryu GH, Kim NKD, Park D, Chung J, Kyung S, Joung JG, Shin HT, Shin SH, Kim Y, Kim BS, Lee H, Kim KM, Kim JS, Park WY, Son DS
Publication: J Mol Diagn, 2017, Vol. 19, Page 651-658
PubMed ID: 28743024 PubMed Review Paper? No
Purpose of Paper
This paper compared next generation sequencing (NGS) quality metrics in frozen and formalin-fixed paraffin-embedded (FFPE) specimens and investigated which metrics were indicators of a pass rate (PR) greater than 80 at the required sequencing depth. The effect of extraction kit on NGS metrics in FFPE specimens was also examined.
Conclusion of Paper
The mean coverage was slightly higher for frozen than FFPE specimens and the pass rate (PR) scores at the required depth (200x for FFPE and 500x for frozen) were higher for frozen than FFPE specimens. The majority of variants detected in frozen specimens had high mean depth and high uniformity >50% (0.8) but there was more variation in mean depth and uniformity >50% in FFPE specimens. The best indicator of a PR500>80 for frozen specimens was the input amount used for hybridization, but the best indicators of PR200>80 for FFPE specimens was a DNA concentration after the pre-PCR step of >26.55 ng/µL and a molality of >3.92 mmol/L after post-PCR. However, in FFPE specimens with a concentration of <26.5 ng/µL after pre-PCR, 86.4% of specimens had a PR200>80 with an average library size of >274 bp.
Although the authors did not specify which extraction kit was which, one kit led to higher mean coverage but less uniformity and lower PR scores than the other kit.
Studies
-
Study Purpose
This study compared NGS quality metrics in frozen and FFPE specimens and investigated what metrics were indicators of a PR>80 at the required sequencing depth. DNA was extracted from 1142 FFPE tumor specimens using the Promega Maxwell 16 CSC DNA FFPE kit and the QIAamp DNA FFPE Tissue kits and from 1283 frozen tumor specimens using QIAamp DNA Mini kits. No details of specimen procurement, processing, or storage were provided. Specimens included chronic myeloid leukemia, melanoma, non-small cell leukemia, colorectal carcinoma, neuroblastoma, small cell carcinoma of the lung, acute myeloid leukemia, breast cancer, thyroid cancer, thymic cancer, basal cell carcinoma, and ovarian cancer. DNA yield was determined by using a Qubit 2.0 fluorometer and spectrophotometry. DNA integrity was determined using a 2200 TapeStation and by real-time PCR amplification. NGS libraries were constructed from 200 ng DNA from frozen specimens and 300 ng DNA from FFPE specimens using SureSelect XT Reagent Kit and sequenced using the HiSeq 2500 sequencing platform. The limit of detection for variants was set at 5% for frozen specimens (>500x) and 10% for FFPE specimen (>200x).
Summary of Findings:
The mean coverage was slightly higher for frozen than FFPE specimens (954x versus 728x), and the PR scores at the required depth (200x for FFPE and 500x for frozen) were higher for frozen than FFPE specimens (97.3% versus 92.9%). Further, there were fewer sites with a uniformity of coverage >50% for frozen specimens than FFPE specimens (0.849 versus 0.876). The majority of variants detected in frozen specimens had high mean depth and high uniformity >50% (0.8), but there was more variation in mean depth and uniformity >50% in FFPE specimens. In specimens with low uniformity, the depth for an individual hotspot rather than the mean depth is critical to understand variant detection. For frozen specimens, DNA input used for hybridization was predictive of a PR500 >80 as this was achieved in 98.9% of specimens when >648 ng of DNA was used for hybridization but only 77.9% if <648 ng was used. For FFPE specimens, DNA concentration after the pre-PCR step was the best indicator of a PR200>80 as 97.6% of specimens has a PR200>80 if the concentration was >26.5 ng/µL versus only 70.9% when the concentration was <26.5 ng/µL. In FFPE specimens with a pre-PCR concentration >26.5 ng/µL, 98% of specimens with >3.92 mmol/L had a PR200>80 versus 70.0% in those with <3.92 mmol/L. For those FFPE specimens with a DNA concentration of <26.5 5 ng/µL after the pre-PCR step, 86.4% of the samples with a library size of >274 bp had a PR200>80 versus 31.0% of the samples with a library size of <274 bp.
Biospecimens
- Tissue - Breast
- Tissue - Colorectal
- Tissue - Thymus Gland
- Tissue - Skin
- Tissue - Thyroid Gland
- Tissue - Ovary
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Lymphoma
- Neoplastic - Melanoma
- Neoplastic - Leukemia
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
Formalin (buffered)
-
Study Purpose
This study compared NGS quality metrics in FFPE specimens extracted using two different kits. DNA was extracted from 1142 FFPE tumor specimens using the Promega Maxwell 16 CSC DNA FFPE kit or the QIAamp DNA FFPE Tissue kits and from two normal colon and stomach specimens using both kits. No details of specimen procurement, processing, or storage were provided. Specimens included chronic myeloid leukemia, melanoma, non-small cell leukemia, colorectal carcinoma, neuroblastoma, small cell carcinoma of the lung, acute myeloid leukemia, breast cancer, thyroid cancer, thymic cancer, basal cell carcinoma, and ovarian cancer. DNA yield was determined by using a Qubit 2.0 fluorometer and spectrophotometry. DNA integrity was determined using a 2200 TapeStation and by real-time PCR amplification. NGS libraries were constructed from 300 ng DNA using SureSelect XT Reagent Kit and sequenced using the HiSeq 2500 sequencing platform. The authors did not specify which kit was which.
Summary of Findings:
Compared to kit A (authors did not specify which kit this was), extraction with Kit B resulted in a higher mean depth (775x versus 728x), higher yields. and smaller differences in cycle threshold (CT) values, but a higher percentage of specimens had a PR500 score >80 (93.7% versus 78.4%) and there was greater uniformity when extraction was with Kit A. DNA purity (260/280 and 260/230 ratios), DNA input, DNA concentration, amount after the pre-PCR step and post-PCR, DNA input for hybridization, average library and insert size, and percentage PCR duplicates were largely comparable between extraction methods. When DNA was extracted from two FFPE normal colon and stomach specimens using the two kits, there was higher mean depth after removing duplicates when Kit B was sued, but there was also more variability in depth and a lower average PR score.
Biospecimens
- Tissue - Breast
- Tissue - Colorectal
- Tissue - Ovary
- Tissue - Stomach
- Tissue - Skin
- Tissue - Thyroid Gland
- Tissue - Thymus Gland
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Leukemia
- Normal
- Neoplastic - Carcinoma
- Neoplastic - Melanoma
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA Automated electrophoresis/Bioanalyzer DNA Fluorometry DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Promega Maxwell 16 CSC DNA FFPE kit
QIAamp DNA FFPE Tissue kit