PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies

The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

► Ig PCR amplification and high throughput sequencing were performed on FFPE tissues. ► Heterogeneous B cell repertoires were detected in all tested reactive lymph node. ► Results of B cell repertoire analyses in frozen and FFPE tissues were close. ► New and old FFPE tissues showed similar results in the B cell repertoire analyses. ► FFPE samples are a “gold mine” for B cell clonal evolution studies.