Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency.
Suppression Subtractive Hybridization (SSH) and its derivative, Pooled Suppression Subtractive hybridization (PSSH), are powerful tools used to study variances larger than ~100 bp in prokaryotic genome structure. The initial steps involve ligating an oligonucleotide of known sequence (the "adaptor") to a fragmented genome to facilitate amplification, subtraction and downstream sequencing. SSH results in the creation of a library of unique DNA fragments which have been traditionally analyzed via Sanger sequencing. Numerous next generation sequencing technologies have entered the market yet SSH is incompatible with these platforms. This is due to the high level of sequence conservation of the oligonucleotide used for SSH. This rigid adherence is partly because it has yet to be determined if alteration of this oligonucleotide will have a deleterious impact on subtraction efficiency. The subtraction occurs when non-unique fragments are inhibited by a secondary self-pairing structure which requires exact nucleotide sequence. We determine if appending custom sequence to the 5' terminal ends of these oligonucleotides during the nested PCR stages of PSSH will reduce subtraction efficiency. We compare a pool of ten S. aureus clinical isolates with a standard PSSH and custom tailed-PSSH. We detected no statistically significant difference between their subtraction efficiencies. Our observations suggest that the adaptor's terminal ends may be labeled during the nested PCR step. This produces libraries labeled with custom sequence. This does not lead to loss of subtraction efficiency and would be invaluable for groups wishing to combine SSH or PSSH with their own downstream applications, such as a high throughput sequencing platform.
Gerrish
Robert S
RS
Departments of Oral Biology, The State University of New York at Buffalo, NY, USA.
Gill
Steven R
SR
D001431:Bacteriological Techniques; D001483:Base Sequence; D003001:Cloning, Molecular; D055028:Comparative Genomic Hybridization / Q000379:methods*; D015342:DNA Probes; D004269:DNA, Bacterial; D018076:DNA, Complementary; D015723:Gene Library; D016680:Genome, Bacterial; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics*
D015342:DNA Probes; D004269:DNA, Bacterial; D018076:DNA, Complementary