PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies |
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Authors: | Hilla Tabibian-Keissar Ginette Schibby Miri Michaeli Aviya Rakovsky-Shapira Noemie Azogui-Rosenthal Deborah K. Dunn-Walters Kinneret Rosenblatt Ramit Mehr Iris Barshack |
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Affiliation: | 1. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel;2. Department of Pathology, Sheba Medical Center, Ramat Gan, Israel;3. King''s College London Medical School, London, UK;4. Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel |
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Abstract: | 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. |
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Keywords: | FFPE tissues, formalin-fixed paraffin-embedded tissues HTS, high throughput sequencing Ig, immunoglobulin MID, multiplex identifier SHM, somatic hypermutation |
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