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Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency

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Abstract

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.

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Abbreviations

SSH:

Suppression subtractive hybridization

PSSH:

Pooled suppression subtractive hybridization

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Acknowledgments

This work was partially funded by start-up funds from the SUNY-Buffalo Center for Excellence in Bioinformatics and Life Sciences, National Institutes of Health 2R01AI059111 (S.R.G.) and T32 DE007034 (R.S.G.).

We thank Vance G. Fowler for providing the clinical isolates used to create the test pool. We thank Gregory Canfield for helpful advice and discussion and Stan Kowalski for encouragement.

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Authors and Affiliations

  1. Departments of Oral Biology, School of Dental Medicine, The State University of New York at Buffalo, Buffalo, NY, USA

    Robert S. Gerrish & Steven R. Gill

  2. Microbiology and Immunology, School of Medicine and Biomedical Sciences, The State University of New York at Buffalo, Buffalo, NY, USA

    Steven R. Gill

  3. Infectious Disease and Genomics, NYS Center of Excellence in Bioinformatics and Life Sciences, The State University of New York at Buffalo, 701 Ellicott Street, Buffalo, NY, 14203, USA

    Robert S. Gerrish & Steven R. Gill

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  1. Robert S. Gerrish
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  2. Steven R. Gill
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Correspondence to Steven R. Gill.

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Gerrish, R.S., Gill, S.R. Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency. Antonie van Leeuwenhoek 98, 573–579 (2010). https://doi.org/10.1007/s10482-010-9465-x

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  • DOI: https://doi.org/10.1007/s10482-010-9465-x

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