Open AccessIsolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High‐Throughput DNA Sequencing
Author(s) -
Xiao Yongli,
Sheng ZongMei,
Taubenberger Jeffery K.
Publication year - 2015
Publication title -
current protocols in microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.813
H-Index - 35
eISSN - 1934-8533
pISSN - 1934-8525
DOI - 10.1002/9780471729259.mc01e08s37
Subject(s) - biology , complementary dna , dna sequencing , rna , host (biology) , dna , cdna library , genetics , computational biology , gene
The vast majority of surgical biopsy and post‐mortem tissue samples are formalin‐fixed and paraffin‐embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9‐year effort by overlapping RT‐PCR from post‐mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high‐throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex‐specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. © 2015 by John Wiley & Sons, Inc.