Salipante Laboratory

Research Program

Our laboratory is in the Department of Laboratory Medicine and Pathology at the University of Washington (Seattle, Washington).  

In addition to pursuing our laboratory's extramural research program, we offer services through the UW Cystic Fibrosis Research Development Program  and Microbial Interactions & Microbiome Center (mim_C).

Our group focuses on the development and application of massively parallel (or "next-generation") DNA sequencing technologies to areas relevant to human health. Our diverse research projects involve both basic science investigation and clinical test development. The purpose of our efforts is to develop and explore new applications of high-throughput sequencing technologies, to translate these technologies to novel clinical diagnostics and basic science discoveries, and to develop bioinformatics tools and data analysis pipelines for these purposes. We aim to learn about the basic biology of human diseases and to apply this new knowledge directly to disease diagnosis, prevention, and treatment.

Our current work is focused on four major, broad areas of translational investigation:

1. Large‑scale sequencing of clinical bacterial isolates.  We have developed methods enabling the whole genome sequencing and analysis of large numbers (hundreds to thousands) of clinical bacterial isolates. These data can be applied to explore many aspects of bacterial infectious diseases, including molecular epidemiology, characterization of the diverse complement of genes that they harbor, and the identification of genes responsible for clinically important properties such as virulence and antibiotic resistance.

2. Detection of low-prevalence cancer mutations. With some modifications, sequencing technologies can now be employed to robustly detect actionable mutations present at very low frequencies (1 in 10,000 to 1 in 100,000 cells). These approaches hold translational opportunities in the sensitive detection of small numbers of cancer cells which persist after therapy (minimal residual disease) and for the early detection of cancers, potentially allowing earlier therapeutic intervention. Reliable detection of very low prevalence mutations also allows basic properties of malignancy to be explored, such the genomic evolution of tumors over time.

3. Development of novel next-generation sequencing technologies and diagnostic tools.  One of our activities is to develop novel applications and methods which utilize next-generation DNA sequencing and leverage its unique properties in various ways. These technologies are applied in translational research activities, and/or are adapted and validated for clinical use in the care of patients.

4. Next-generation methods for assessing Microsatellite Instability. Microsatellite instability (MSI) is a molecular tumor phenotype resulting from genomic hypermutability. The spontaneous gain or loss of nucleotides from microsatellite tracts is the diagnostic hallmark of MSI and manifests as novel alleles of varying length. Although MSI holds well-defined implications for therapeutic choices, prognostic assessment, and risk appraisal for hereditary cancer predisposition syndromes in patients afflicted with colorectal cancer, MSI represents a relatively recent discovery in cancers it remains not fully understood, and has newfound importance with the advent of immune checkpoint inhibitor immunotherapies (such as PDL-1 and PD-1 inhibitors). We have developed methods to infer MSI events using massively parallel, or “next-generation”, DNA sequencing technologies, which now enable MSI to be interrogated with a breadth and quantitative precision not previously achievable. We have demonstrated the superiority of our approach to existing clinical methods of MSI diagnosis, and have established several clinical assays at using that methodology.  In applying our methods to examine MSI events on a genome-wide scale, we have uncovered many novel surprises about the nature of MSI, including the existence of cancer-specific patterns of instability events, identification of MSI positive tumors across most cancer types examined, and recognition of MSI as a continuous, rather than discrete, phenotype. 

P. aeruginosa Mutant Library

With financial support from the Cystic Fibrosis Foundation, the Salipante Lab has assumed responsibility for distributing clones from the ordered P. aeruginosa Mutant Library developed by Dr. Colin Manoil's laboratory.   In publications using these isolates, please acknowledge financial support from the Cystic Fibrosis Foundation (Grants # SINGH19R0 and SINGH24R0).   Measuring the impact of the P. aeruginosa mutant library in this way is very important for our grant renewals. 

Strains from the arrayed P. aeruginosa PAO1 Two-Allele Transposon Mutant Library are available for a charge to the research community through a nonprofit cost center at the University of Washington. The strains have been single colony-purified, and there are two mutants available for most nonessential genes. In-depth information about the library can be found in the following publications: Jacobs et al. 2003, PNAS 100:14339 and Held et al. 2012, J. Bacteriol. 194:6387. The accompanying the mutants available can be found in the PA two-allele library mutant manifest, linked below.

Instructions for Requesting individual mutants 

Please understand that we are a small academic laboratory and require the cooperation of requestors when we are fulfilling requests, especially for international requests that require import or other permits. The PA two-allele library mutant manifest provides information about each mutant in the two-allele library.  Most of the column headings are self-explanatory.  There is also a description of column headings on the second sheet of the file (“Legend”).  We apologize, but as a small research lab, we cannot send out replacements if our transposon location in a mutant cannot be confirmed or has been mis-assigned.  We do appreciate knowing confirmation status and will update our manifest file with the results as an aid to future users.  When selecting mutants from the library, please also consider carefully what control strains you will require for your experiments (see Control strains and Strain variation due to mexT mutations, below) and be sure to request these strains with your individual mutants.
To request individual mutants:

1. Download and complete a copy of the order form (Excel file) that corresponds to your type of institution (either academic or nonacademic, links provided under "Order Forms", below), and complete all required fields (shown in yellow) .  There is an instruction sheet included on the order form, but please let us know if you have any questions about the form itself, the mutants, or the ordering process.  At  present, purchase orders are required for all transactions, and a valid Purchase Order number must be provided on the order form.  Please note that currently, our institution only supports payment by check, wire transfer, or ACH.

2. On your order form, please be sure to provide a FedEx account number if possible.  If you are unable to provide a FedEx account number, we can bill you for shipping, but institutional fees will be charged on top of base shipping costs.  If you elect to be billed for shipping, please contact us before placing your order so that you can include the appropriate fees in your Purchase Order.

3. Download and complete a Customer Profile form, if you have not previously submitted a request to our group.  

4. Send the completed forms to stevesal[ a t ] uw.edu.

For requests from countries requiring import or other permits, the requestor must obtain the necessary permits and email copies to stevesal [at] uw.edu, and assumes responsibility for resolving questions or concerns raised by customs officials during transit.  All charges resulting from failure to provide the required permits or to resolve outstanding customs issues will be paid by the requestor, including the cost of return shipment following customs rejection.   

Order Forms and Manifests

• Academic institution order form

• Non-academic institution order form

• PA two-allele library mutant manifest (Excel spreadsheet) PA Orfs correspond to locus tags assigned in the PAO1 reference genome

• Additional information about the PA Two allele Library

• Customer Profile form Please complete and return this with your order form, if you have not previously submitted a request to our group.

Choice of strains

In creating a large arrayed mutant library like this one, it is inevitable that some assignments will fail to check out. In addition, high-throughput growth and distribution may lead to some mixed cultures.
We have done our best to minimize cross-contamination and insertion mis-assignment by colony purification and two rounds of sequencing of insertion location (but not absence of the corresponding wild-type gene).

• If we have obtained two or more Sanger sequencing reads corresponding to within 500bp of the called insertion site, there is a "+" in the Sanger confirmed column.  

• If we were able to detect the exact sequence of the called insertion site via Tn-seq performed on a pool of the entire library, there is a "+" in the Tn-seq confirmed column.  

• If we were able to detect an insertion site within 100bp of the called insertion site via Tn-seq of the library pool, and the Sanger read that was used to call the location was not an exact read there is a "+" in the Tn-seq confirmed column.  

Basically, Tn-seq confirmations suggest the transposon insertion site exists in the library but it does not say it is in the assigned well, whereas Sanger sequencing is done on specific wells.  We therefore believe our highest quality insertion locations to be those confirmed by both Sanger and Tn-seq.
We have included more than one insertion for most genes, and suggest that multiple mutants corresponding to genes of interest be requested to help provide coverage in case individual mutants cannot be confirmed. Unfortunately, we are unable to provide replacements or refunds for mutants that cannot be confirmed.

Receipt and maintenance of strains

Individual strains are sent as stab cultures in semisolid agar (ie, agar slants).  After receiving a mutant, it is important that a sample of the strain be maintained as a frozen stock (–80°C) in the recipient laboratory. We recommend that the researcher streak from the stab onto a nutrient medium such as LB agar (without antibiotic) immediately after receipt, then scoop up a generous sample from the dense part of the streak for the frozen stock (in LB containing 5% DMSO v:v).
Quality control is performed to ensure that you are sent a viable stock. It is possible that a strain you requested is viable for only a short period of time due to the mutation it harbors, and would not be recoverable after shipping. Once a strain has been shipped and is at that time viable, we are unable to provide refunds or reshipment without an additional order being placed.

Confirmation of strains

We also urge investigators to check the identities of mutants by PCR or sequencing prior to use.  We recommend that researchers test at least 10 isolated colonies of each strain by PCR both with flanking primers and with a transposon-specific primer paired with a flanking primer to confirm mutants.
For each gene of interest, the researcher should design flanking primers or utilize the primers (designed by Mike Jacobs at the University of Washington Genome Center) that are listed in the PA two-allele library mutant manifest. The primers are computer-generated and have not been tested by us. They should be tested initially using the wild type parent strain to verify production of an appropriate wild-type band. For insertion strains, the same PCR should yield either no band or a band corresponding to a very large product.
To demonstrate the presence of the transposon insertion, use a transposon-specific primer with one of the flanking primers.  The transposon used in each strain is listed in column K of the library Excel file.  If the transposon used was ISphoA/hah, then use primer Hah minus 138 (5’-cgggtgcagtaatatcgccct-3’), and if the transposon was ISlacZ/hah, use lacZ 148 (5’-gggtaacgccagggttttcc-3’). The transposon primer should be used in conjunction with one of the flanking primers according to the orientation of the transposon relative to the gene.  There is additional information about this procedure in the Additional information about the PA Two allele Library file, linked below.
On occasion, purified transposon mutant strains produce both wild-type and insertion mutant bands in the PCRs described above. We suspect these are usually bacteria carrying both wild-type and insertion mutant alleles and arise from transposon insertion in one copy of a tandem duplication that includes the gene of interest. Such strains are frequently found for essential genes in which wild-type function is required for viability.

When you have either confirmed or been unable to confirm a mutant strain, please send an email to stevesal [at] uw.edu to let us know your results.

Control strains and Strain variation due to mexT mutations

There are variations in the mexT gene commonly seen for PAO1 strains, including the subline MPAO1 and the resulting mutants in our transposon mutant library. The variations arise because MPAO1 and mutants of it carry an inactivating mutation in the mexS gene, which leads to constitutive activity of the MexT regulator. The constitutive MexT activity slows growth and reduces survival of frozen strains, and (different) mexT-minus mutations can thus be enriched in frozen stocks of mutants or during strain passaging. The mexT- minus mutants can be distinguished from mexT-plus strains because they form somewhat larger colonies and are more sensitive to chloramphenicol (tested on LB agar containing 10 micrograms/mL chloramphenicol) and nalidixic acid (tested on LB supplemented with 64 micrograms/mL nalidixic acid). Several papers describing this variation include: 

• Luong PM, et al. Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF. Journal of Bacteriology. 2014;196(2):504-513. doi:10.1128/JB.01050-13

• Lee et al, Reconstructing a wild-type Pseudomonas aeruginosa reference strain PAO1. Journal of Bacteriology. 2021; 203(14):e00179-21 doi:10.1128/JB.00179-21.

Strain LPAO,  constructed by Lee et al., is a derivative of MPAO1 in which the inactive mexS allele has been reverted, restoring this regulatory system to its functional state.  We recommend that users ascertain their phenotype of interest in both MPAO1 and LPAO, in order to rule out possible contributions from mexT variation.

Pooled transposon mutant library

Our P. aeruginosa transposon mutant pooled library was made by saturation-level transposition in MPAO1.  To achieve high insertion density, we used a transposon with low insertion specificity derived from Tn5.  To limit false assignments caused by polarity, the transposon carried an outward-facing promoter designed to express downstream genes.  Mutants were selected on LB containing 60mg/mL tetracycline.  In our Tn-seq, the pool was found to contain approximately 110,000 unique insertion sites. More information can be found in PNAS. 2015 112(16):5189. doi: 10.1073/pnas.1422186112. Pools are sold in 1-mL aliquots with a titer of more than 10^9 cells/mL.  Pricing is shown on the relevant order form.

DISTRIBUTION OF THE POOLED P. AERUGINOSA LIBRARY IS NOT BEING SUPPORTED AT THIS TIME.

Ordering complete arrayed library copies and pooled libraries.

Complete arrayed library and pooled library orders may be initiated either by contacting us at stevesal [at] uw.edu or by filling out the Excel order form (instructions are on the first sheet) and emailing the form to stevesal [at] uw.edu.  Current pricing is shown on the relevant order form (academic or nonacademic).

In making replicates of the entire library for distribution, several steps of quality control are performed.  Strains are assessed for their growth by visual assessment of turbidity, and strains that did not grow well are grown up individually and included in supplemental plates.  Sanger sequencing is performed on a subset of wells (~3% of the library) to ensure plate orientation and library integrity (typically <2% of mutants provide unexpected sequence).

DISTRIBUTION OF THE COMPLETE, ARRAYED P. AERUGINOSA LIBRARY IS NOT BEING SUPPORTED AT THIS TIME.

Reporting Mutant Names and Genotypes

In publications, please reference strains from the Two-Allele Library by strain name (PW####). Refer to the genotype in the following way: gene name (or PAORF if there is no gene name)-well name (final three digits of the location field) as the allele number ::Transposon name (ISphoA/hah or ISlacZ/hah).  For example, for strain PW1001, the genotype is recF-B10::ISphoA/hah, and for strain PW1003, the genotype is PA0005-E05::ISlacZ/hah. 

Acknowledgements/citations in publications using this resource

For publications resulting from the use of these strains please acknowledge the following:

1) Please reference Held et al. 2012, J. Bacteriol. 194:6387 as the source of the strains

2) Please acknowledge financial support from the Cystic Fibrosis Foundation (Grants SINGH19R0 and SINGH24R0), which maintains the isolate collection.  Measuring the impact of the P. aeruginosa mutant library through the publication record is very important for our grant renewals. 

Current Fees

• Academic users - $30 per strain ($210 minimum order = 7 strains, although fewer strains may be requested)

• Non-academic users - $50 per strain ($500 minimum order = 10 strains, although fewer strains may be requested)

• Additional fees include material costs, labor, and shipping  (shipping costs are only applied if recipient's FedEx account # is not supplied)

• A 15.6% institutional overhead fee is added to all orders

• Our institution only supports payment by check, wire transfer, or ACH at this time.  

Publications

Publications from the Salipante Lab can be found through PubMed or Google Scholar.  

Protocols

Detailed protocol for ultrasensitive detection of low-prevalence variation using single molecule molecular inversion probes (smMIPs).  

Follow this link for a detailed worksheet and a protocol detailing the use of smMIPs to detect extremely low-prevalence somatic variation (1:10,000 to 1:60,000 mutant alleles).  Analysis software can be found at: ssh://git@bitbucket.org/uwlabmed/smmips_analysis.git

If you use these protocols in your work, please cite the paper from which this protocol originated:

Waalkes, K. Penewit, B.L. Wood, D. Wu, S.J. Salipante. 2017. Ultrasensitive detection of acute myeloid leukemia minimal residual disease using single molecule molecular inversion probes.  Haematologica. 2017 Sep;102(9):1549-1557. doi: 10.3324/haematol.2017.169136. Epub 2017 Jun 1. PMID: 28572161