Alternative Fixative for Genome Friendly Tissue Handling to Implement Genomic Analysis in the 100,000 Genomes Project


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Clare Craig1,Emily Shaw2,Rachel Nelan3,Sally Smith4,Noor Jahan4,Victoria Hipperson4,Faisal Mihaimeed4,Shirley Henderson1,Miao He5,Zoya Kingsbury5,Lisa Browning6,Margaret Ashton-Key7,Afshan Siddiq1,Emma Walsh1,Kay Lawson1,Martina Mijiskovic1,Alona Sosinsky1,Greg Elgar1,Joanne Mason1,Tom Fowler1,James Brenton8,Mark Ross5,Louise Jones9
1Genomics England,2University Hospital Southampton NHS Trust,3Queen Mary University,4Barts Health NHS Trust,5Illumina,6Oxford Health NHS Foundation Trust,7Southampton University Hospital NHS Trust,8Cancer Research UK's Cambridge Institute,9Genomics England; Queen Mary University London and Barts Health NHS Trust

Abstract

Background

The 100,000 Genomes Project is a Government funded initiative to create the infrastructure for genomic medicine in the NHS. It aims to sequence 100,000 genomes on patients with rare disease or cancer. For patients with cancer, tumour samples are required for sequencing together with germline DNA extracted from blood. Traditionally tissue samples are placed in formalin fixative to maintain morphology for histological analysis, however, this damages DNA and makes the interpretation of whole-genome sequences (WGS) unreliable. This study analysed the quality of DNA and WGS generated from tissues using an alternative fixative and assessed the feasibility of introducing this into routine practice.

Method

Biopsy-sized tissue samples were taken from surgical excision specimens from patients who had consented for use of their tissue in research. The tissue biopsies were placed in formalin (FFPE) or the alternative fixative PAXgene®, and matched samples were snap-frozen in liquid nitrogen (FF). Tissues were processed; H&E sections assessed for morphology, and DNA was extracted for WGS on the Illumina platform.

Results

Morphology was compared between samples FF or fixed in formalin or PAXgene®: the two fixatives gave comparable morphology which was superior to the FF tissue. DNA yields were similar for all sample conditions, but pre-sequencing QC metrics were poorer for FFPE samples. WGS showed good coverage uniformity in the PAXgene® samples, comparable to the FF samples, whilst coverage was uneven in the FFPE samples with many unique variants introduced due to the fixation.

Conclusion

PAXgene® fixed tumour samples give WGS of comparable quality to FF tissue without the generation of numerous unique variants which make FFPE samples unsuitable for reliable WGS. The ease of use of PAXgene® compared to FF tissue makes it more suitable for wider-scale implementation in the clinical setting.