Technical in-depth comparison of two massive parallel DNA-sequencing methods for formalin-fixed paraffin-embedded tissue from victims of sudden cardiac death.
Author(s): Adolfsson E, Qvick A, Gréen H, Kling D, Gunnarsson C, Jonasson J, Gréen A
Publication: Forensic Sci Int Genet, 2021, Vol. 53, Page 102522
PubMed ID: 33945952 PubMed Review Paper? No
Purpose of Paper
This paper compared sequencing metrics between libraries constructed using the HaloPlex and Twist kits from DNA isolated from postmortem formalin-fixed paraffin-embedded (FFPE) cardiac tissue. Additional libraries were prepared from blood using HaloPlex.
Conclusion of Paper
All FFPE HaloPlex and Twist libraries were successfully sequenced. HaloPlex libraries had a higher sequencing depth despite a higher percentage of duplicate reads, shorter average read length, lower percentage of bases with 20% coverage and less uniform coverage than the Twist libraries. Importantly, 15 genes lacked coverage in HaloPlex blood libraries; however, when libraries were constructed from blood DNA using Twist, only one gene lacked complete coverage. Percentage coverage at 20% and average sequencing depth of the HaloPlex libraries were higher for FFPE specimens with moderately (>1000 bp) fragmented DNA compared to those with severely fragmented DNA (<1000 bp), but DNA fragmentation did not affect the percentage of bases with 20% coverage and average sequencing depth of Twist libraries. Libraries from severely fragmented FFPE specimens also had elevated GC content, although the bias was larger when Twist was used compared to HaloPlex. The sensitivity of FFPE HaloPlex and Twist libraries for variant detection in blood specimens were 93.5% and 97.8%, respectively. The majority (69/85) of missed variants in sequenced HaloPLex libraries generated from the FFPE specimen were due to poor coverage.
Studies
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Study Purpose
This study compared sequencing metrics between libraries constructed using the HaloPlex and Twist kits from DNA isolated from postmortem FFPE cardiac tissue. Additional libraries were prepared from blood using HaloPlex. Eight FFPE heart tissues were obtained from an archive. Heart specimens had been obtained during autopsy of donors that died as a result of a sudden cardiac event 6 years prior to the study (no additional details were provided). For each patient, blood specimens were sequenced with HaloPlex as part of the autopsy (no further details were provided). DNA was extracted from FFPE sections using the QIAamp DNA FFPE Tissue Kit, quantified using Quant-it dsDNA BR Assay Kit, and fragment length was evaluated using Genomic DNA Screen Tape. DNA libraries were constructed using the HaloPlex HS for FFPE tissue Kit and the Twist Kit. For specimens with moderately fragmented DNA (>1000 bp) the protocol was optimized by using the Twist Kit in conjunction with the Westburg NGS kit, while the Westburg noFrag DNA Kit was used for severely degraded DNA (<1000 bp). Twist libraries were sequenced using NextSeq, HaloPlex and miSeq.
Summary of Findings:
All FFPE HaloPlex and Twist libraries were successfully sequenced, but the Haloplex libraries had a higher percentage of duplicate reads and a shorter average read length than the Twist libraries (14.4-49.2% versus 5.6-10%, and 91 versus 131 bp). Despite a higher sequencing depth for HaloPlex compared to Twist libraries (408–1565X, vs 57–171X, P < 0.001), Twist libraries had a higher percentage of bases with 20% coverage Twist (85.8-99.5% versus 76.19-99.44%) and Twist more uniform coverage. Importantly, 15 genes lacked coverage in HaloPlex blood libraries; however, when libraries were constructed from blood DNA using Twist, only one gene lacked complete coverage. The percentage of bases with 20% coverage and the average depth of HaloPlex libraries were higher for FFPE specimens with moderately fragmented DNA (>1000 bp) than those with severely (<1000 bp) fragmented DNA (98.55-99.44% versus 76.19-90.44% and 749-1565 X versus 408-771 X); however, DNA fragmentation did not affect the percentage of bases with 20% coverage or the average depth of Twist libraries. The number of reads from Twist libraries was also not correlated with mean DNA length, DNA integrity number (DIN), or library insert size. Libraries from severely fragmented FFPE specimens also had elevated GC content but the bias was larger when Twist was used compared to HaloPlex. A total of 1226 and 1300 variants were detected in HaloPlex libraries from FFPE and blood specimens, respectively, with 1215 shared among FFPE and blood libraries that resulted in a sensitivity of 93.5% for the FFPE specimen. The majority (69/85) of missed variants in the sequenced HaloPLex libraries from the FFPE specimens were due to poor coverage and, as expected, sensitivity was higher for moderately fragmented than severely fragmented specimens. Using Twist libraries from FFPE specimens, 1295 variants were detected and 1271 of those variants were shared with the sequenced HaloPlex library from blood with a false negative rate of 2.2% and a false positive rate of 1.8%.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
- Cardiovascular Disease
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Blood
Heart tissue
Next generation sequencing Specific Technology platform TWIST
HaloPlex