[Six-sequence-tagged site (STS) versus eight-STS scheme for detection of Y chromosome microdeletions].

OBJECTIVE: To compare the six-sequence-tagged site (STS) with the eight-STS scheme in the detection of Y chromosome microdeletions. METHODS: Using real-time quantitative PCR, we compared the results of the six-STS (sY84, sY86, sY127, sY134, sY254, sY255) scheme with those of the eight-STS (sY84, sY86, sY127, sY134, sY254, sY255, sY145, sY152) scheme in detecting Y chromosome microdeletions. RESULTS: No statistically significant difference was found in the detection rate of the deletion of the azoospermia factor (AZF) regions between the six-STS and eight-STS methods (9.34% ï¼»575/6177ï¼½ vs 8.85% ï¼»542/6122ï¼½, P > 0.05). CONCLUSION: Though the eight-STS scheme increased the detection of AZFd, its detection rate of the AZF region deletion was not significantly different from that of the six-STS method. From the perspectives of experimental operation, economic cost and clinical strategy guidance, the six-STS is better than the eight-STS scheme for the detection of Y chromosome microdeletions.

Molecular characterization of the Yp11.2 region deletion in the Chinese Han population.

The Y chromosome is male-specific and is important for spermatogenesis and male fertility. However, the Y chromosome is poorly characterized due to massive palindromes and inverted repeats, which increase the likelihood of genomic rearrangements, resulting in short tandem repeats on the Y chromosome or long fragment deletions. The present study reports a large-scale (2.573~2.648 Mb) deletion in the Yp11.2 region in a Chinese population based on the analysis of 34 selected Y-specific sequence-tagged sites and subsequent sequencing of the breakpoint junctions on the Y chromosome from 5,068,482-5,142,391 bp to 7,715,462-7,716,695 bp. The results of sequence analysis indicated that the deleted region included part or all of the following five genes: PCDH11Y, TSPY, AMELY, TBL1Y, and RKY. These genes are associated with spermatogenesis or amelogenesis and various other processes; however, specific physiological functions and molecular mechanisms of these genes remain unclear. Notably, individuals with this deletion pattern did not have an obvious pathological phenotype but manifested some degree of amelogenesis imperfecta.

Generation of recombinant influenza virus bearing strep tagged PB2 and effective identification of interactional host factors.

The genome of influenza A virus is negative-sense and segmented RNA, which is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp) during the virus life cycle. The viral RdRp is thought to be an important host range and virulence determinant factor, and the 627 site of PB2 subunit is a highly acceptable key site of RdRp function. Besides, the function of RdRp is modulated by several host factors. Identification of the host factors interacting with RdRp is of great interest. Here, we tried to explore an effective method to study virus-host interaction by rescuing replication-competent recombinant influenza viruses carrying Strep tagged PB2. Subsequently, we tested several biological characteristics of recombinant viruses in cells and pathogenicity in mice. Then, we purified of protein complex of Strep tagged PB2 and host factors of interest from 293 T cells infected with recombinant viruses. After purification, we performed mass spectrometry to identify these proteins that interacting with PB2. We identified 57 host factors in total. Through Gene Ontology (GO) and Protein-Protein interaction (PPI) network analysis, we revealed the function and network of these proteins. In summary, we generated replication-competent recombinant influenza viruses by inserting a Strep-Tag into PB2 and purified host factors interacting with viral RdRp bearing a 627 K or 627E PB2. These proteins might function as host range and virulence determinants of influenza virus.

Elucidating host-microbe interactions in vivo by studying population dynamics using neutral genetic tags.

Host-microbe interactions are highly dynamic in space and time, in particular in the case of infections. Pathogen population sizes, microbial phenotypes and the nature of the host responses often change dramatically over time. These features pose particular challenges when deciphering the underlying mechanisms of these interactions experimentally, as traditional microbiological and immunological methods mostly provide snapshots of population sizes or sparse time series. Recent approaches - combining experiments using neutral genetic tags with stochastic population dynamic models - allow more precise quantification of biologically relevant parameters that govern the interaction between microbe and host cell populations. This is accomplished by exploiting the patterns of change of tag composition in the microbe or host cell population under study. These models can be used to predict the effects of immunodeficiencies or therapies (e.g. antibiotic treatment) on populations and thereby generate hypotheses and refine experimental designs. In this review, we present tools to study population dynamics in vivo using genetic tags, explain examples for their implementation and briefly discuss future applications.

Deletion of b1/b3 shows risk for expanse of Yq microdeletion in male offspring: Case report of novel Y chromosome variations.

RATIONALE: This study aimed to report 1 family case with novel Y chromosome structural variations by an established next-generation sequencing (NGS) method using unique STSs. PATIENT CONCERNS: The case studied was from a family with a father and son (the proband). G-band staining was used for karyotype analysis. Y chromosome microdeletions were detected by sequence-tagged site (STS)-PCR analysis and a new NGS screening strategy. DIAGNOSES: Semen analysis showed that the proband was azoospermic. The patient had an abnormal karyotype (45,X[48%]/46,XY[52%]). His father exhibited a normal karyotype. STS-PCR analysis showed that the proband had a deletion of the AZFb+c region, and his father had no deletion of STS markers examined. The sequencing method revealed that the patient had DNA sequence deletions from nt 20099846 to nt 28365090 (8.3 Mb), including the region from yel4 to the Yq terminal, and his father exhibited a deletion of b1/b3 and duplication of gr/gr. INTERVENTIONS: The proband was advised to undergo genetic counseling, and consider the use of sperm from a sperm bank or adoption to become a father. OUTCOMES: The proband was azoospermic. AZFc partial deletions may produce a potential risk for large AZFb+c deletions or abnormal karyotypes causing spermatogenic failure in men. LESSONS: The NGS method can be considered a clinical diagnostic tool to detect Y chromosome microdeletions. The partial AZFc deletions and/or duplications can be a risk of extensive deletions in offspring.

Cross-transferability of SSR markers developed in Rhododendron species of Himalaya.

Rhododendron is a genus of evergreen woody ornamental plants of northern hemisphere with strong cold resistance, attractive flowers and high altitude adaptation capacity. The genus originated and diversified from Sino-Himalayan region and spread across the world, and has high species diversity in Northeast India. To assess cross-species amplification, we tested 32 microsatellites markers in fifteen taxa of the genus Rhododendron of North-eastern Himalaya, of which fourteen microsatellites were newly developed from Rhododendron simsii, and eighteen microsatellites were previously developed from Rhododendron catawbiense and Rhododendron mucronatum var. ripense. Nine pairs of primers were amplified successfully in all species, however, none of them was failed for amplification in any of the species. The average observed heterozygosity, expected heterozygosity and PIC value were recorded as 0.310, 0.433 and 0.379 respectively. Clustering based on neighbour-joining analysis revealed the potential of these markers to segregate species according to their subgenus level, however, subspecies exhibited closeness with each other. Cross-application of these microsatellite loci will provide a potentially useful tool to investigate the genetic diversity, population structure, gene flow, phylogenetics and evolutionary relationships in species of genus Rhododendron.

Prevalence of Y chromosome microdeletion in azoospermic infertile males of Iraqi population.

In human gamete development, the important period is spermatogenesis, which is organized by specific genes on Y chromosome. In some cases, the infertile men have shown microdeletions on Y chromosome, which seemed as if the structural chromosome variance is linked to the reduction of sperm count. This study aimed to determine the frequency and patterns of Y chromosome microdeletions in azoospermia factor (AZF) of Iraqi infertile males. Here, 90 azoospermic infertile males as a study group and 95 normal fertile males as control group were investigated for the microdeletion of AZF loci using numerous sequence-tagged sites. Of these 90 infertile male patients, 43 (47.8%) demonstrated Y chromosome microdeletions, in which AZFb region was the most deleted section inazoospermia patients (33.3%) followed by deletions in the AZFc region (23%), while there were no microdeletion in the AZFa region. The largest microdeletion involved in both AZFb and AZFc was detected in six azoospermic patients (6.7%). The present study demonstrated a high frequency of Y chromosome microdeletions in the infertile Iraqi patients which is not reported previously. The high frequency of deletions may be due to the association of ethnic and genetic factors. PCR-based Y chromosome screening for microdeletions has a potential to be used in infertility clinics for genetic counselling and assisted reproduction.

Identification of QTL for resistance to root rot in sweetpotato (Ipomoea batatas (L.) Lam) with SSR linkage maps.

BACKGROUND: Sweetpotato root rot is a devastating disease caused by Fusarium solani that seriously endangers the yield of sweetpotato in China. Although there is currently no effective method to control the disease, breeding of resistant varieties is the most effective and economic option. Moreover, quantitative trait locus (QTL) associated with resistance to root rot have not yet been reported, and the biological mechanisms of resistance remain unclear in sweetpotato. Thus, increasing our knowledge about the mechanism of disease resistance and identifying resistance loci will assist in the development of disease resistance breeding. RESULTS: In this study, we constructed genetic linkage maps of sweetpotato using a mapping population consisting of 300 individuals derived from a cross between Jizishu 1 and Longshu 9 by simple sequence repeat (SSR) markers, and mapped seven QTLs for resistance to root rot. In total, 484 and 573 polymorphic SSR markers were grouped into 90 linkage groups for Jizishu 1 and Longshu 9, respectively. The total map distance for Jizishu 1 was 3974.24 cM, with an average marker distance of 8.23 cM. The total map distance for Longshu 9 was 5163.35 cM, with an average marker distance of 9.01 cM. Five QTLs (qRRM_1, qRRM_2, qRRM_3, qRRM_4, and qRRM_5) were located in five linkage groups of Jizishu 1 map explaining 52.6-57.0% of the variation. Two QTLs (qRRF_1 and qRRF_2) were mapped on two linkage groups of Longshu 9 explaining 57.6 and 53.6% of the variation, respectively. Furthermore, 71.4% of the QTLs positively affected the variation. Three of the seven QTLs, qRRM_3, qRRF_1, and qRRF_2, were colocalized with markers IES43-5mt, IES68-6 fs**, and IES108-1 fs, respectively. CONCLUSIONS: To our knowledge, this is the first report on the construction of a genetic linkage map for purple sweetpotato (Jizishu 1) and the identification of QTLs associated with resistance to root rot in sweetpotato using SSR markers. These QTLs will have practical significance for the fine mapping of root rot resistance genes and play an important role in sweetpotato marker-assisted breeding.

SNP markers for low molecular glutenin subunits (LMW-GSs) at the Glu-A3 and Glu-B3 loci in bread wheat.

The content and composition of seed storage proteins is largely responsible for wheat end-use quality. They mainly consist of polymeric glutenins and monomeric gliadins. According to their electrophoretic mobility, gliadins and glutenins are subdivided into several fractions. Glutenins are classified as high molecular weight or low molecular weight glutenin subunits (HMW-GSs and LMW-GSs, respectively). LMW-GSs are encoded by multigene families located at the orthologous Glu-3 loci. We designed a set of 16 single-nucleotide polymorphism (SNP) markers that are able to detect SDS-PAGE alleles at the Glu-A3 and Glu-B3 loci. The SNP markers captured the diversity of alleles in 88 international reference lines and 27 Mexican cultivars, when compared to SDS-PAGE and STS markers, however, showed a slightly larger percent of multiple alleles, mainly for Glu-B3. SNP markers were then used to determine the Glu-1 and Glu-3 allele composition in 54 CIMMYT historical lines and demonstrated to be useful tool for breeding programs to improve wheat end product properties.

Karyotypic abnormalities and molecular analysis of Y chromosome microdeletion in Iranian Azeri Turkish population infertile men.

Infertility is one of the major health-threatening problems in communities which may lead to psychological problems among couples. Y chromosome abnormalities and microdeletions have recently been considered as one of the male infertility factors. The aim of this study was to evaluate different chromosomal disorders and azoospermia factor b (AZFb), AZFc and AZFd microdeletions in idiopathic non-obstructive oligo or azoospermia infertile men. One hundred infertile (78 azoospermia and 22 oligospermia) and 100 fertile men were included in this study. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were evaluated by electrochemiluminescence. Karyotyping was performed according to standard methods and interpreted using the International System for Human Cytogenetic Nomenclature (ISHCN) recommendation. For Y chromosome microdeletion analysis, a multiplex polymerase chain reaction (PCR) was performed using STS primers. Higher FSH (24.32 ± 15.32 versus 8.02 ± 3.37, p < 0.0001) and LH (14.97 ± 8.26 versus 5.42 ± 2.73, p < 0.0001) were observed in infertile patients compared to their fertile counterpart. Additionally, 14% of infertile patients exhibited abnormal karyotype. The  frequency of Y chromosome microdeletions in azoospermic and oligospermic patients was 32.05% (25/78) and 0% (0/22), respectively. Additionally, in azoospermic patients, the highest microdeletion frequency was related to the AZFc region (80%). Our data indicate the presence of chromosomal changes in the most infertile men, suggesting karyotype and molecular analysis of Y chromosome microdeletions for genetic counseling before assisted reproduction.Abbreviations: ART: assisted reproductive technology; AZF: azoospermia factor; DAZ: deleted in azoospermia; FCS: fetal calf serum; FSH: follicle stimulating hormone; LH: luteinizing hormone; PCR: polymerase chain reaction; SRY: sex-determining region Y; STS: sequence-tagged sites.

Simultaneous Detection of Multiple Pathogenic Targets with Stem-Tagged Primer Sets.

Simultaneous multiple gene detection is indispensable for the detection of various genes in a small sample obtained by an invasive method. A typical detection method is probe-based fluorescence melting curve analysis by means of real-time PCR. It is very limited because, for each target, a probe sequence with at least a different Tm must be designed. To overcome this limitation, we developed a simultaneous multiple gene detection method based on a giant amplicon molecular beacon. PCR was performed by attaching stem sequences with different Tm values to each primer set, and the melting Tm was measured by hybridizing the stem sequences at both ends of the amplified amplicon; this generated well-separated Tm signals. The important point here is that the stem sequence that produces the Tm signal is an arbitrarily selectable sequence unrelated to the target gene. Because it is arbitrarily selectable, the desired Tm can be freely adjusted. As a result, we succeeded in the simultaneous detection of four samples with the use of only one fluorophore. Theoretically, a combination of five fluorophores could detect more than 20 multiple genes simultaneously.

Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients.

The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen's kappa coefficient of 0.85 with 95% CI = 0.72-0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90-0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

GLADS: A gel-less approach for detection of STMS markers in wheat and rice.

Sequence tagged microsatellite site (STMS) are useful PCR based DNA markers. Wide genome coverage, high polymorphic index and co-dominant nature make STMS a preferred choice for marker assisted selection (MAS), genetic diversity analysis, linkage mapping, seed genetic purity analysis etc. Routine STMS analysis involving low-throughput, laborious and time-consuming polyacrylamide/agarose gels often limit their full utility in crop breeding experiments that involve large populations. Therefore, convenient, gel-less marker detection methods are highly desirable for STMS markers. The present study demonstrated the utility of SYBR Green dye based melt-profiling as a simple and convenient gel-less approach for detection of STMS markers (referred to as GLADS) in bread wheat and rice. The method involves use of SYBR Green dye during PCR amplification (or post-PCR) of STMS markers followed by generation of a melt-profile using controlled temperature ramp rate. The STMS amplicons yielded characteristic melt-profiles with differences in melting temperature (Tm) and profile shape. These characteristic features enabled melt-profile based detection and differentiation of STMS markers/alleles in a gel-less manner. The melt-profile approach allowed assessment of the specificity of the PCR assay unlike the end-point signal detection assays. The method also allowed multiplexing of two STMS markers with non-overlapping melt-profiles. In principle, the approach can be effectively used in any crop for STMS marker analysis. This SYBR Green melt-profiling based GLADS approach offers a convenient, low-cost (20-51%) and time-saving alternative for STMS marker detection that can reduce dependence on gel-based detection, and exposure to toxic chemicals.

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol.

Analysis of the length polymorphisms in sequence-tagged-site sY1291 on Y chromosome in Vietnamese men of infertile couples.

This study aims to analyze the length polymorphisms in sequence-tagged-site (STS) sY1291 of the Y chromosome in Vietnamese men of infertile couples. All 322 DNA samples were amplified with the sY1291 primer by the quantitative fluorescent polymerase chain reaction (QF-PCR) assay. DNA sequencing technique was employed to evaluate the accuracy of QF-PCR results. The study showed 273 out of 322 DNA samples had the presence of STS sY1291, accounted for 84.78%. The QF-PCR results showed that there were various lengths in STS sY1291: 507 bp, 512 bp, 523 bp and 527 bp. The most prevalent length in STS sY1291 was 507 bp (87.5%), the others were 512 bp (4.8%), 523 bp (4.8%) and 527 bp (2.9%). We found that the observed length polymorphisms derived from differences in the number of mononucleotide Thymine (T) repeats in its structure. It stretched from 22 T to 39 T. DNA sequencing results identified that the number of mononucleotide T repeats causes these polymorphisms. However, the pair-wise alignment between the obtained and reference sequence was 77%. It can be seen that the length polymorphisms in STS sY1291 observed in QF-PCR results was accurate but it is still difficult to sequence fragments with mononucleotide repeats.

An oligo-His-tag of a targeting module does not influence its biodistribution and the retargeting capabilities of UniCAR T cells.

Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution.

Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.

A Backcross Line of Thatcher Wheat with Adult Plant Leaf Rust Resistance Derived from Duster Wheat has Lr46 and Lr77.

The widely grown hard red winter wheat cultivar Duster released in 2006 has remained highly resistant to leaf rust caused by Puccinia triticina in the southern Great Plains of the United States. In contrast, many of the winter wheat cultivars in this region are susceptible to leaf rust. The goal of this study was to identify the number and chromosome location of leaf rust resistance genes in a line of Thatcher*2/Duster wheat that was selected for adult plant leaf rust resistance. The Thatcher*2/Duster line was crossed with Thatcher (Tc) and a recombinant line inbred line (RIL) population was advanced to the F6 generation by single-seed descent. The parents and RIL population were phenotyped for leaf rust resistance in three field plot tests and in an adult plant greenhouse test. Single-nucleotide polymorphism (SNP) markers derived from the Illumina Infinium iSelect 90K wheat SNP array, kompetitive allele-specific polymerase chain reaction assays on chromosome 3BL, and a sequence tagged site (STS) marker on chromosome 1BL were used to construct a genetic map of the RIL population. The STS marker csLV46G22 that is linked with resistance gene Lr46 on chromosome 1BL, and SNP marker IWB10344 that is linked with Lr77 on chromosome 3BL, were significantly associated with lower leaf rust severity. Duster has at least three adult plant resistance genes for leaf rust resistance because it was previously determined to also have the adult plant resistance gene Lr34. Duster is a valuable source of durable leaf rust resistance for hard red winter wheat improvement in the Great Plains region.

Delimitation of wheat ph1b deletion and development of ph1b-specific DNA markers.

KEY MESSAGE: We detected the deletion breakpoints of wheat ph1b mutant and the actual size of the deletion. Also, we developed ph1b deletion-specific markers useful for ph1b-mediated gene introgression and genome studies. The Ph1 (pairing homoeologous) locus has been considered a major genetic system for the diploidized meiotic behavior of the allopolyploid genome in wheat. It functions as a defense system against meiotic homoeologous pairing and recombination in polyploid wheat. A large deletion of the genomic region harboring Ph1 on the long arm of chromosome 5B (5BL) led to the ph1b mutant in hexaploid wheat 'Chinese Spring,' which has been widely used to induce meiotic homoeologous recombination for gene introgression from wild grasses into wheat. However, the breakpoints and physical size of the deletion remain undetermined. In the present study, we first anchored the ph1b deletion on 5BL by the high-throughput wheat 90K SNP assay and then delimited the deletion to a genomic region of 60,014,523 bp by chromosome walking. DNA marker and sequence analyses detected the nucleotide positions of the distal and proximal breakpoints (DB and PB) of the ph1b deletion and the deletion junction as well. This will facilitate understanding of the genomic region harboring the Ph1 locus in wheat. In addition, we developed user-friendly DNA markers specific for the ph1b deletion. These new ph1b deletion-specific markers will dramatically improve the efficacy of the ph1b mutant in the meiotic homoeologous recombination-based gene introgression and genome studies in wheat and its relatives.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.