Single Molecule Next Generation Analytics

My laboratory was one of the first to embrace a genome-wide systems approach to directly address whether we identified the exception or the rule to the biological questions we tackle.  This included the development of chromosome-specific aCGH and genome-wide 454 sequencing capabilities in collaboration with the Joint Genome Initiative as we transitioned through the Illumina Hi-Seq technologies and have now moved to single-molecule analytics. Recently we developed three new tools to establish the quality of sperm RNA and RNA prepared from Frozen Formalin Paraffin-Embedded clinical samples.  First, we developed REDa, the RNA Element Detection algorithm.  This turned the problem of fragmentation, which is often observed in sperm, into an advantaged solution based on the concept of RNA elements that we developed.  RNA elements are defined as contiguous transcript segments mapping back to the genome.  They also resolve as a series of previously unannotated isoforms of known transcripts and orphans within known and novel regulatory regions (1).  Second, we developed a novel computational approach to quantify the integrity of sperm RNAs.  This algorithm grades the quality of every sample based on a set of RNAs typically retained as full-length transcripts and consistently found present in human sperm (2,3).  The third novel tool we developed based on our previous studies (4) determines a sample's cellular purity.  All transcripts discovered from elements are assessed as a proportion of transcripts in each tissue type reported in GTeX (1) or a similar database.  As part of my laboratory's longstanding tradition, all software and sequence data are publicly available.      

​​​​REFERENCES

  1.  Estill, M.S., Hauser, R. Krawetz, S.A. (2019) RNA element discovery from germ cell to blastocyst. Nucleic Acids Research. 18:2263-2275 PMID: 30576549   
  2. Jodar, M., Sendler, E., Moskovtsev, S. Librach, C., Goodrich, R., Swanson, S., Hauser, R., Diamond, M. and Krawetz, S.A. (2016) Response to comment on Absence of sperm RNA elements correlates with idiopathic male infertility. Science Translational Medicine. 8:353tr1 PMID: 26157032
  3. Sendler, E., Johnson, G.D., Mao, S., Goodrich, R.J., Diamond, M.P., Hauser, R., and Krawetz, S.A. (2013)Stability, Delivery and Functions of Human Sperm RNAs at Fertilization.  Nucleic Acids Research 41:4104-4117. PMID: 23471003
  4. Martins, R.P., Leach, R.E. and Krawetz, S.A. (2001) Whole body gene expression by data mining.  Genomics 72:34-42.  PMID: 11247664