BACKGROUND
current genome-wide analyses mainly depend on either gene expression profiling or large-scale mutant phenotyping .
expression profiling allows for the detection of changes in gene expression levels; however, the pattern of gene expression does not always reflect gene function.
for example, in a genome-wide analysis of the budding yeast saccharomyces cerevisiae gene deletion mutant set, less than 7% of the genes that showed increased mrna expression in response to four different conditions were required for growth under the same conditions, and deletion of some of the most highly expressed genes had no effects on cell proliferation.
in addition, many genes necessary to maintain normal cell fitness under these treatments did not have significantly altered expression levels  <cit> .
large-scale mutant phenotyping monitors changes in mutant fitness or other visible traits, and provides direct assessment of the requirements of genes under specific conditions.
the availability of the open reading frame  deletion mutant collections in the budding yeast s. cerevisiae and the fission yeast schizosaccharomyces pombe has proven to be advantageous in this approach.
the principal challenge in large-scale phenotyping is distinguishing individual mutants.
the current method of choice is to tag each mutation with a unique dna sequence called a “barcode”  <cit> .
because barcode tags are part of the mutations, the proportion of an individual barcode reflects the proportion of that barcode-associated mutant in a population.
thus, following barcode frequencies can identify mutants with a desired growth phenotype from a population of diverse mutants, an approach referred to as parallel analysis  <cit> .
an advantage of parallel analysis of barcoded mutants is that mutations causing deleterious or weak phenotypes can be efficiently detected.
for example, to identify virulence genes in salmonella typhimurium, mice were infected with a s. typhimurium mutant library where each mutant carried a unique  <dig> bp barcode tag.
the barcodes that were lost from the post-infection population identified the genes required for virulence  <cit> .
this approach has also been extended to cultured human cells using barcode-tagged cdna and shrna libraries to discover genes whose overexpression and down-regulation could suppress cancer cell growth and survival  <cit> .
in all three screens, the mutants of interest diminished in the population but could be revealed by detecting the loss of their associated barcodes by dna arrays  <cit> .
the budding yeast s. cerevisiae is the model organism where barcode-tagged mutagenesis has been the most successfully exploited  <cit> .
its small, sequenced and well-annotated genome and the efficient gene deletion techniques allowed the production of a collection of complete orf deletion mutants where each deletion mutant is tagged by two unique barcodes  <cit> .
the barcodes can be amplified en masse by pcr to generate probes for commercially available high-density microarrays to take a “census” of the relative abundance of each mutant in a culture under a variety of conditions  <cit> .
two independent genetic screens using this barcode-tagged deletion mutant set have identified many genes whose deletion caused lengthened chronological lifespan as detected by an increase in the abundance of the long-lived mutant barcodes  <cit> .
as long-lived mutants and mutants with normal lifespan are often morphologically indistinguishable, the barcode approach to monitor the length of lifespan of many mutants in parallel demonstrated the power of parallel analysis for detecting weak phenotypes.
s. pombe is an important model system that has many of the advantages of s. cerevisiae, including a sequenced genome and well-established microbiological, genetic and molecular biological approaches  <cit> .
unlike s. cerevisiaes.
pombe shares a number of similar features with mammals including rna interference, aspects of rna splicing and the requirement for the mitochondrial genome for survival of wild type cells  <cit> .
a barcode-tagged deletion strain set for s. pombe, in which each deletion mutant carries two barcodes, has also been developed recently  <cit> .
most of the mutants in the s. pombe and s. cerevisiae orf deletion sets are null mutations, and haploid mutants lacking essential genes are not present.
truncated essential proteins with partial or altered function and mutated alleles of essential genes with reduced expression levels have been shown to produce viable and detectable phenotypes such as changes in transcriptional silencing and lifespan  <cit> .
therefore, a mutant collection generated by a different approach that includes mutations that impair the function of essential and non-essential genes would be beneficial for parallel analysis of phenotypes and genome-wide screens.
an alternative mutagenesis method in s. pombe is non-homologous recombination-dependent integration of a selectable marker  <cit> , which can generate a wide variety of mutations.
insertions in the coding sequences can produce truncated proteins with either no or altered function.
insertions in the 5’ and 3’ region of a gene can change protein expression levels by compromising promoter function and mrna stability, respectively  <cit> , and produce viable mutations in essential genes  <cit> .
previous studies in s. pombe characterized two classes of transformants when cells were transformed with linear dna that had limited or no homology to the genome  <cit> .
one class of transformants contained single or tandem copies of linear dna stably inserted in the genome, and the insertion events were often accompanied by deletions in the integrated dna vectors and adjacent genome sequences.
the distribution of insertions in the genome appeared random  <cit> .
the other class, which constituted the majority of the transformants, maintained the transfected dna as unstable, extrachromosomal circular dna despite the absence of a known origin of dna replication in the vector  <cit> .
chua et al.
<cit>  and davidson et al.
<cit>  used this approach to create random s. pombe mutants by transforming a ~ <dig>  kb dna fragment containing the ura4+ gene into strains with the ura4-d <dig> mutation, a deletion that removes the sequences homologous to the  <dig>  kb ura4+ dna from the genome.
these studies revealed two important features.
first, only one insertion event was identified in each mutant by the criteria of southern blotting.
thus, it is possible to generate a library of transformants where each individual contains a single mutation  <cit> .
second, many of the transformants contained insertions composed of multiple full or partial copies of the vector at each site.
the complex structure of these insertions made mapping their genomic locations challenging  <cit> .
to facilitate genome-wide functional analysis in s. pombe, we generated a barcode-tagged s. pombe library of random insertion mutants that retains features equivalent to the budding and fission yeast orf deletion mutant collections, enables identification of mutants in the absence of knowing the barcode sequences, and allows parallel analysis with basic molecular biology techniques.
we created an insertion dna cassette composed of the ura4+ selectable marker, a random barcode, a “buffer” sequence to protect the barcode from degradation during integration, and other sequences to allow modifications of the integrated vector.
of the ~ <dig>  insertion mutations generated, phenotypic analysis and mapping of a subset indicated that the insertions were broadly distributed in the genome.
thus, this work demonstrates the feasibility and potential utility of constructing barcoded, random insertion libraries in s. pombe, and provides approaches that can allow rapid analysis of large collections of barcode-tagged mutations in other organisms.
RESULTS
a linear dna vector loses sequences from the ends during non-homologous recombination-mediated insertion in s. pombe
as a first test of using insertion mutagenesis to generate barcode-tagged insertion mutations in s. pombe, an initial barcoded insertion vector was made by pcr amplification of the ura4+ gene using ura4+-specific primers to produce a ura4+ gene with a random barcode sequence at its 3’ end .
cells with the ura4-d <dig> mutation were transformed with the linear vector dna and maintained on minimum medium without uracil to select for ura+ transformants.
anticipating that the majority of the transformants would carry extrachromosomal ura4+ circles, the initial transformants were re-grown twice on non-selective medium to allow cells to lose unstable ura4+ circles.
these colonies were then replica-plated onto both selective medium and minimum medium plates with 5-fluoroorotic acid  to identify transformants with stably integrated ura4+, which are inviable in 5-foa medium  <cit> .
the initial test with  <dig> ura+ transformants identified eight that were sensitive to 5-foa, indicating that only about 4% of the transformed cells contained stably integrated dna.
the rest of the transformants  likely carried the ura4+ marker extrachromosomally and had lost the circular ura4+ dna upon growth on 5-foa-containing medium.
analysis of the eight ura4+ insertion mutants by pcr revealed that seven mutants had lost their barcodes, indicating that successful integration was frequently accompanied by deletion of barcodes.
these results are consistent with previous observations of low frequency stable integration by non-homologous recombination and deletion on the ends of the inserted dna  <cit> , underscoring the need to increase the proportion of stable transformants and to prevent deletion of barcodes.
construction and characterization of the bacterial barcode-tagged insertion dna library
the final insertion dna vector contains a barcode between a “buffer sequence” and the 5’ end of the ura4+ gene so deletions would first occur in the buffer sequence or the selectable marker before altering the barcode .
this buffer sequence included new sequence elements with additional potential functions: a modified  <dig> bp λ dna sequence  at the 5’ end to prevent the degradation of the barcode, followed by a modified human hsp <dig> promoter that contains a lexa binding site  <cit> , and a mutated loxp site named lox <dig>  <cit> .
the lexa-hsp <dig> promoter can be bound by any lexa fusion protein and may stimulate transcription through the λ dna sequences, which have been modified to contain no atg sequences, and would translate the first atg in s. pombe genomic dna  <cit> .
the lox <dig> site can, in the presence of cre recombinase, recombine with the lox <dig> site to allow integration  of a plasmid  <cit> .
the new barcode consists of a 27-nucleotide random sequence that is interrupted at specific positions with  <dig> a bases.
this “interrupted barcode” is flanked by two sfi i restriction sites so that the barcodes can amplified by pcr, digested with sfi i restriction endonuclease and oligomerized in a head-to-tail orientation for sequencing several barcodes in a single reaction .
as a test of this approach, a fragment containing the barcode dna and flanking insertion vector sequences was first pcr amplified and then digested with sfi i. gel-purified barcode monomers were then ligated together to form oligomers .
a ladder of barcode oligomers was generated, and cloning and sequencing of those oligomers longer than five barcodes confirmed that they were ligated in a head-to-tail manner.
we have successfully cloned and sequenced up to  <dig> tandem barcodes per plasmid, and could routinely clone an average of seven barcodes per plasmid in pilot experiments .
thus, when sampling a population of random mutants, each sequenced plasmid provides information on about seven different barcodes and sequencing  <dig> to  <dig> plasmids can provide a sufficiently large sample size to identify barcodes that are present at high frequency.
as the barcodes used in this work were designed with  <dig> random bases, they provide a total of  <dig> or  <dig>  ×  <dig> possible barcode sequences.
we generated a barcode library of  <dig>  ×  <dig> bacterial clones from  <dig> sub-libraries containing  <dig>  ×  <dig> to  <dig>  ×  <dig> clones per sub-library.
each sub-library contains independent and different random barcodes and helps preserve the high complexity of the library.
our goal was to generate  <dig>  fission yeast mutants with unique and non-redundant barcodes, which should give a 76% chance to cover all annotated genes .
in general, the number of fission yeast mutants produced was ≤ 1% of the total number of barcode clones present in the bacterial sub-libraries used for mutagenesis, so all mutants have a ≥ 95% chance of having unique barcodes.
construction of the fission yeast barcode-tagged insertion mutant library
the  <dig>  kb linear barcoded insertion vector dna was purified from the bacterial barcode sub-libraries after digestion with bamh i enzyme and used to generate the initial transformants.
to enrich the overall proportion of stable integrants, we tested the possibility of utilizing low concentrations of 5-foa to select against cells bearing multiple copies of extrachromosomal ura4+ circles.
cells bearing ura4+ circles are expected to have multiple ura4+ genes per cell.
higher levels of ura <dig> protein should produce more toxic 5-fluorouracil in the presence of low concentrations of 5-foa, causing these cells to have a growth disadvantage .
in contrast, stable integrants with one or few copies of integrated ura4+ dna do not produce as much ura <dig> protein, and are less sensitive to the same treatment .
to test this assumption, fission yeast cells transformed with the linear insertion vector dna were immediately plated on low-dose 5-foa selective medium plates .
the resulting transformants were verified for stable integration by growing them on normal-dose 5-foa medium  and minimum medium plates without uracil.
an initial test on  <dig> transformants grown on low-dose 5-foa medium plates identified  <dig> transformants which died when transferred to medium with normal-dose 5-foa plates.
this result indicates 30% of the transformants contained stably inserted vector dna, a significant enrichment of mutants with stable integration compared to the previously observed 4%.
the low-dose 5-foa selection procedure was used in large-scale generation of fission yeast insertion mutants .
transformants obtained on low-dose 5-foa selective medium plates and passing the first verification on normal-dose 5-foa medium plates were transferred to non-selective medium in 96-well plates.
each four 96-well plates were then used to make one 384-colony array on a selective medium and a normal-dose 5-foa omni plate to confirm the ura+ and 5-foa-sensitive phenotypes of stable insertion mutants.
mutants behaving as true stable insertion mutants were stored individually in 384-well plates .
in addition, five ~ <dig> -mutant pools  and one ~ <dig> mutant pool  were made for a total of ~ <dig>  mutants.
the insertion mutant library contains diverse mutations
to evaluate the diversity of mutations in the insertion mutant library, four genetic screens were performed on  <dig> mutants from ten 384-well plates for phenotypes that could be easily scored.
in the first screen, we looked for mutants that grew slowly or could not grow on minimum medium plates supplemented with only adenine, leucine and histidine to complement the original auxotrophic mutations in our strain.
we discovered  <dig> mutants containing mutations that impeded growth enough to prevent visible colony formation on minimal medium.
based on the gene ontology database -predicted mutant frequency  <cit>  and the fact that only ~57% of the s. pombe genome contains protein coding genes in which mutations are more likely to generate detectable phenotypes than in intergenic regions  <cit> ,  <dig> auxotrophic mutants would be expected in the  <dig> insertion mutants assayed .
because not every mutation will generate a visible phenotype, the frequency of auxotrophic mutants in this assay was consistent with the go prediction.
<dig> %
a. the estimated number is from amigo database  <cit>  by searching genes using keywords “amino acid biosynthesis” and “nucleobase biosynthesis”, and excluding genes involved in the adenine, histidine, leucine and uracil pathways as the parental strain in defective in these pathways and all mutants are ura+.
b. total gene number in s. pombe genome  =  <dig>
c. the estimated number is calculated as “the total number of mutants tested ” × “amigo expectation” × “the fraction of protein coding sequences in the total s. pombe genome ”.
d. not determinable; white colony color could also result from mitochondrial defects  <cit> .
e. the estimated number is from kegg  <cit>  .
f. not determinable; no associated terms or categories in amigo or kegg.
in the second assay, we selected for mutations that altered colony color on low adenine medium.
as the parental yeast strain of the insertion library mutants contains the ade6-m <dig> mutation, we screened for an alteration in colony color from pale pink  to red or white when cells were grown in rich medium with low concentrations of adenine.
mutations in only two genes, ade6+ and ade7+, are known to cause red pigment accumulation in cells.
one mutant out of the  <dig> screened showed dark red colony color on low adenine medium plate and was identified as an ade7- insertion mutant .
thirteen mutants which turned white on low adenine medium plates were also isolated.
although this phenotype can be associated with mutations in the adenine biosynthesis pathway that produce substrates for the ade <dig> and ade <dig> enzymes, mutants with compromised mitochondrial function have also been reported to demonstrate a similar phenotype  <cit> .
in the third test, we screened for temperature-sensitive mutations that allow mutant cells to grow normally at 30°c, but not at 36°c.
a total of  <dig> such mutants that grew slowly or could not form visible colonies at 36°c were recovered .
mutations that cause temperature-sensitive growth are not well-characterized, so these data cannot be used to estimate the expected mutant frequency.
however, the isolation of temperature-sensitive mutants does demonstrate the wide variety of mutations present in the library.
in the fourth experiment, we selected for mutations that confer cellular resistance to ethidium bromide .
etbr toxicity is primarily due to inhibition of the circular mitochondrial genome.
as with mammalian cells, s. pombe requires a functional mitochondrial genome for survival so etbr is highly toxic.
if s. pombe and mammalian cells acquire certain nuclear mutations, the so-called rho <dig> cells devoid of mitochondrial dna can be generated by long term selection in etbr-containing medium with specific supplements  <cit> .
using this etbr selection procedure, we identified  <dig> strains that are etbr-resistant .
to determine whether these mutant phenotypes were caused by the ura4+ insertion or mutations induced by transformation, we crossed the mutants to a wild type strain and performed tetrad analysis on a subset of the identified mutants to verify co-segregation of the ura4+ marker and the phenotypes.
of  <dig> strains examined,  <dig> showed 2: <dig> segregation of ura4+ in  seven tetrads .
by this criteria, the majority of mutants contained single insertions.
of the  <dig> mutants that showed slow or no growth on minimum medium, the ura4+ marker co-segregated with the phenotype in a 2: <dig> ratio in  <dig> mutants, indicating that most of the mutant phenotypes were linked to the insertion mutations.
similar results were observed in mutants exhibiting altered color on low adenine medium .
of the  <dig> temperature-sensitive mutant assayed, one was sterile, four showed poor spore viability and three of the remaining six showed 2: <dig> co-segregation with ura4+ .
the source of the unlinked mutations is unknown but may be due to the mutagenic effects of transformation  <cit> , and underscores the requirement of validating mutants from premade collections by recreating the mutation in a new strain to determine if the phenotype is regenerated.
the sum of these data show that the majority of these insertions contain the ura4+ vector in a single locus and the insertion locus is genetically linked to the mutation in most of these mutants.
mapping of insertion sites and analysis of the structures of inserted vectors
previous studies on non-homologous recombination-mediated mutagenesis in s. pombe showed that the genomic locations of insertion vectors were difficult to determine by inverse pcr  <cit> .
we therefore tested two previously established methods for large-scale insertion site determination, thermal asymmetric interlaced -pcr  <cit>  and splinkerette-adaptor pcr  <cit> .
tail-pcr uses alternating high and low annealing temperatures, a set of arbitrary degenerate  primers and three nested insertion dna-specific primers to amplify a small region of insertion dna and the adjacent genomic sequence   <cit> .
tail-pcr only detected genome-insertion junctions in a portion of mutants analyzed .
in some mutants, only repetitive insertion vector sequences were amplified, indicative of tandem integration of vector dna in these mutants .
in other mutants, mitochondrial dna was found co-integrated with insertion vector .
these results indicate that these additional dna fragments provided binding sequences for the degenerate or vector-specific primers .
therefore, we also used splinkerette pcr, which involves ligating a double strand dna adaptor to genomic dna fragments digested with restriction enzymes that cut frequently in the genome but not in the vector   <cit> .
the resulting products provide templates for pcr amplification of neighboring genomic dna using specific primers on the splinkerette adaptor and insertion vector.
the splinkerette approach produced pcr fragments of defined size and some genomic sequences, allowing the estimation of the region of the insertion in some mutants and detection of insertion-chromosome junction in others .
as the splinkerette approach also gave vector or mitochondrial sequences in some mutants, we pursued additional mapping approaches.
we adapted splinkerette and inverse pcr to a new method, inverse splinkerette pcr, which may eliminate extra copies of tandem insertion vector in some mutants before pcr .
in inverse splinkerette pcr, genomic dna from insertion mutants was digested with a restriction enzyme that cuts once  in the insertion vector and frequently in the genome .
the digestion generates fragments with one chromosomal dna end and one insertion vector end, which can be brought together by intramolecular ligation to produce circular dna.
digestion of the ligated dna with sfi i enzyme linearizes circular dna composed of truncated 5’ part of the insertion vector and a genomic fragment, and generates a unique overhang on the λ buffer of vector for ligating splinkerette adaptor.
the genomic dna bordered by the partial vector dna and splinkerette adaptor can be amplified by specific primers on insertion vector and splinkerette.
this method can reveal the ecor v site closest to the insertion site and together with the length of the pcr product, to generate an approximate location of the insertion mutation .
in cases where the ecor v site is very close to the insertion site, one may detect the precise junction sequences between chromosomes and insertion vector.
we also directly cloned the genomic sequences flanking the insertion vector in e. coli by the lox66/lox <dig> integration system .
as the insertion mutations are all marked with the mutated loxp sequence, lox <dig>  introducing a plasmid bearing the lox <dig> sequence into cells expressing cre recombinase should allow stable integration of the lox <dig> plasmid into the lox <dig> sequence.
we therefore transformed the plasmid plox <dig>  which has the lox <dig> sequence and a selectable marker for g <dig> resistance in yeast and kanamycin resistance in e. coli, into s. pombe insertion mutant cells bearing the prep <dig> plasmid with or without the cre recombinase gene.
because plox <dig> does not contain a functional yeast replication origin, integration into the genome is required for its stable inheritance and g <dig> resistance in yeast cells.
following introduction of plox <dig>  cells expressing cre recombinase produced more g418-resistant colonies than cells without cre, indicating that cre promotes efficient integration of plox <dig> into the genome .
specific integration of plox <dig> to lox <dig> of the insertion vector was verified by pcr using primers on plox <dig>  and the insertion vector .
while expected pcr products were generated from cre-expressing cells, the same pcr reactions using those g418-resistant cells without cre did not yield bands corresponding to plox <dig> integration, indicating these cells may bear plox <dig> extrachromosomally .
sequencing of pcr products from cells expressing cre also confirmed that they contained the recombined lox66/lox <dig> hybrid and the wild type loxp sequences .
to clone genomic dna flanking the insertion sites, genomic dna of g418-resistant, plox66-integrated cells was digested with spe i and xba i, and ligated under conditions favoring intramolecular ligation.
the presence of the puc origin on plox <dig> allowed us to clone genomic sequences neighboring the insertion site in e. coli for sequencing .
using this method, we cloned and verified the insertion site of the mutant 18_m <dig> .
using tail-pcr, we initially characterized  <dig> mutants, of which we were able to determine the genomic sequences at one end of the insertion sites in  <dig> mutants.
in  <dig> mutants, only repetitive insertion vector sequences were amplified and sequenced, and mitochondrial dna was found co-integrated with insertion vector in eight mutants .
we have also used inverse splinkerette pcr to map insertions in  <dig> mutants and determined approximate location of the insertion vector in  <dig> mutants, nine of which we were not able to determine by tail-pcr .
these results suggest that although tail-pcr and inverse splinkerette pcr are not sufficient for mapping all insertion sites, they are complimentary to each other in deciphering insertion positions accompanied by different insertion structures.
splinkerette pcr and lox66/71-based cloning also allowed the determination of insertion sites .
among the  <dig> mutants in which exact or approximate insertion locations were determined,  <dig> of them have insertion mutation on chromosome  <dig>  with  <dig> on chromosome  <dig> and  <dig> on chromosome  <dig>  in the  <dig> mutants where at least one end was mapped to a defined locus,  <dig> were located in orfs or non-coding rna genes and  <dig> were found in intergenic regions.
in the seven mutants in which both ends of the insertion sites were characterized, deletions of genomic sequences  were found in only three mutants , and deletions of a wide range of sizes, from  <dig> bp to  <dig>  kb, were observed at ends of the insertion vectors in all mutants.
tandem integration of multiple insertion vectors were detected in all but one mutant  in head-to-tail, head-to-head or tail-to-tail orientations.
the structures of the insertion vectors in these mutants is consistent with the ends of some linear vector dna being resected by nucleolytic activities in cells and ligated together before being integrated as tandem copies into the genome as previously observed by others .
discussion
in this study, we created a fission yeast insertion mutant library in which all mutants were tagged with unique barcode sequences and stored as two readily available selection platforms.
the 384-well mutant arrays allow genetic screens on individual mutants and can be extended to genetic approaches such as synthetic genetic array   <cit> .
these mutant arrays have been used to identify mutants with four distinct phenotypes  as well as strains that are hyper-sensitive to cancer chemotherapeutics camptothecin and bleomycin .
in addition to 384-well mutant arrays, mutant pools of  <dig> mutants are available for parallel analysis.
the insertion mutagenesis used in this study relied on random non-homologous recombination, where a vast majority of transformants have unstable, circularized vector dna and only a small portion have stable insertions.
to facilitate the collection of stable insertion mutants, we included low-dose 5-foa in our initial selecting medium as an effort to eliminate unstable cells bearing high copy number of ura4+ vector and producing high levels of ura4p , and subsequently re-screened for mutants that were stably ura4+ and 5-foa-sensitive .
while this approach increased the proportion of stable insertion mutants among the total transformants , we note that some mutants might have been excluded.
for example, insertions into genomic regions where expression switches between on and off states  would be excluded from the final library.
likewise, insertion at a locus that causes high ura4+ expression or tandem integration of many functional ura4+ markers could result in increased sensitivity to low-dose 5-foa and eliminate some mutants during the initial selection.
to increase the versatility of the mutant library, some previously characterized functional dna sequences were included in our insertion vector, including the lox <dig> sequence and the mutated human hsp <dig> promoter.
we demonstrated that the mutated lox <dig> and lox <dig> could undergo cre recombinase-dependent integration in s. pombe, similar to what has been reported in mammalian cells  <cit> , and showed that this method can be used to clone genomic sequences surrounding the insertion mutations using the plox <dig> plasmid.
the mutated human hsp <dig> promoter, which exhibits dramatically reduced activity in s. pombe, was tethered with a lexa binding site for its potential activation by a lexa dna-binding domain and transactivator fusion protein.
while the goal was to provide an opportunity to ectopically express nearby genes, the tandem integrations of insertion vector and co-integration of mitochondrial dna could impede the utilization of this promoter to activate genes near the insertion site.
testing of the hsp <dig> promoter was, therefore, not pursued.
another novel addition to this insertion mutant library is the inclusion of unique barcodes.
these barcodes allow one to take a census of the selected mutants by tracking barcode frequencies after the selection.
barcode sequencing can be facilitated by converting individual sfi i site-bordered barcodes to barcode oligomers , which allows for the generation of multiple barcode sequences per sanger sequencing reaction, providing a cost-effective and rapid alternative compared to cloning and sequencing individual barcodes.
although the high-throughput sequencing or microarray is possible analytic means for our mutant library, the design of our barcodes provides a medium-throughput alternative which requires only easily accessible laboratory techniques.
moreover, in contrast to the necessity of sophisticated bioinformatics support for microarray and high-throughput sequencing, the sequences of several hundred barcodes obtained from our approach can be easily sorted and analyzed with basic spreadsheet software.
we note that our oligomerization and sequencing strategy can also be used to monitor other nucleic acids in cells .
by amplifying a small region around a sequence difference between two different nucleic acids, our oligomerization and sequencing method can easily produce ~ <dig> or more sequences, which should be sufficient for determining the relative proportions of the two forms with data that are much easier to process than data from a high-throughput sequencing experiment.
random insertion mutant libraries may overcome some challenges in the study of essential genes.
mutants that lack essential genes are not present in haploid gene deletion mutant banks such as the budding and fission yeast orf deletion collections.
among the s. pombe insertion mutants analyzed in this work, we found that in six mutants, the insertions were identified in or adjacent to essential genes , presumably generating truncated proteins or altering the expression of these genes.
these results indicate that this insertion mutant library approach provides opportunities for functional analysis of essential genes.
the insertion events characterized in this library are consistent with those shown in previous studies, including insertions in both genes and intergenic regions, large deletions of insertion dna and little or no deletions of surrounding chromosomal sequences .
we also discovered  <dig> mutants with mitochondrial dna co-integrated with the insertion vector.
the presence of mitochondrial dna in the wild type s. pombe nuclear genome has recently been characterized with one wild type strain containing  <dig> mitochondrial dna insertions in its nuclear chromosomes  <cit> .
mitochondrial dna fragments were also found in all repaired plasmid-based double strand breaks in s. pombe cells in an independent study  <cit> .
it is worth noting that capture of mitochondrial dna in the nuclear genome has also been observed in hemiascomycetous yeasts, plant, insect, rodent and human cells and appears to be an active and ongoing process  <cit> , indicating that s. pombe transformation provides a way to study this process.
one consequence of mitochondrial and tandem ura4+ dna insertions is that they can impede the detection of the insertion sites by tail-pcr.
we have tested three additional approaches for mapping insertion mutations: splinkerette pcr, inverse splinkerette pcr and lox66/71-dependent cloning.
while these methods were not 100% efficient, we showed that they could complement each other in defining insertion sites.
one advantage of tail-pcr is that it detects the junction of insertion vector and genomic sequences and provides the exact location of the integrated vector.
in contrast to tail-pcr, inverse splinkerette pcr in our mutants directly determined the closest ecor v sites to the 5’ end  of insertion vector.
together with the length of the pcr products, only approximate regions of insertion could be obtained.
while we did not follow up results from our inverse splinkerette pcr, one could determine the exact location of insertion by cloning the pcr product and sequencing the genomic regions with gene-specific primers.
depending on the complexity of insertion structure, splinkerette pcr could determine the insertion vector-chromosome junction or the closest restriction sites used in the assay to the insertion site.
it is important to note that all mutants are tagged by the lox <dig> sequence, which allows the cloning of the genomic sequences flanking the insertion in e. coli in the event that insertion mutations could not be mapped by these three pcr methods.
in addition to non-homologous recombination-based integration, other methods for generating insertion mutations include transposon-mediated mutagenesis.
at least three types of transposons have been analyzed in a genome-wide context in s. pombe.
the s. pombe retrotransposon tf <dig> has been shown to exhibit preference for targeting the promoters of rna polymerase ii transcribed genes  <cit> .
the piggybac  transposon, originally isolated from cabbage looper moth, preferably targets ttaa sites in the genome.
although as much as 79% of transposition events of piggybac  analyzed in a haploid s. pombe strain was located in intergenic regions, it was assumed that this seemingly preference of pb transposon for intergenic sequences was a consequence of selective pressure on insertions in orfs that cause reduced fitness  <cit> .
high throughput sequencing performed in both studies indicated that the transposition events of both transposons broadly distribute among the three chromosomes.
the hermes transposon from housefly musca domestica has strong preference for t at position  <dig> and for a at position  <dig> of the target sites and has been adapted for s. pombe.
the limited number of insertion mutants analyzed in that study suggested that hermes targets both intergenic regions and coding sequences with no apparent bias  <cit> .
insertions generated by non-homologous recombination in our work also have a broad distribution on the three chromosomes, similar to what was observed in tf <dig> retrotransposon and pb transposon  <cit>  and a previous report on non-homologous recombination in s. pombe <cit> .
although 60%  of the partially characterized insertions resided in orfs or non-coding rna genes, the enrichment of this type of insertions may be due to pre-selection of the corresponding mutants by visible phenotypes.
thus, the three transposon approaches and our insertion vector approach can create a wide variety of mutations.
our approach has the advantage of adding unique barcodes to each insertion, which has not been applied to the transposon approaches.
the main difference between non-homologous integration and transposon transposition is the structures of insertion events.
while transposons generally integrate at individual genomic locations as unmodified single copies with defined junctions between genomic and transposon dna, tandem integration of the insertion vector dna or co-insertion of non-nuclear dna during non-homologous recombination-based integration make this junction more variable.
the simple insertion events in transposon mutagenesis allow for high-throughput sequencing for mapping insertion sites, while the complex insertion structures generated by non-homologous recombination require insertion sites to be determined by low/medium-throughput approaches.
nonetheless, the high mutation variety, the presence of random barcodes, and the availability of multiple methods for mapping insertion mutations still make this insertion mutant library an attractive tool for genome-wide studies that can complement the existing s. pombe orf deletion set.
CONCLUSIONS
as an effort to further support genome-wide studies in s. pombe, we generated a barcode-tagged s. pombe insertion mutant library which is available as pools of mixed mutants for parallel analysis and in the form of 384-well mutant arrays for genetic screens on individual mutants.
the four genetic screens conducted in this work indicate that the library has a wide variety of mutations and is suited for the selection for diverse phenotypes.
the design of the barcode tags allows detection and quantification of the barcodes with readily available molecular biological techniques, and does not require prior knowledge of the barcodes and mutations in the mutant strains to conduct genetic screens.
the barcode-tagging strategy described here can be easily adapted to other model systems.

 current genome-wide analyses mainly depend on either gene expression profiling or large-scale mutant phenotyping. expression profiling allows for the detection of changes in gene expression levels; however, the pattern of gene expression does not always reflect gene function. for example, in a genome-wide analysis of the budding yeast saccharomyces cerevisiae gene deletion mutant set, less than 7% of the genes that showed increased mrna expression in response to four different conditions were required for growth under the same conditions, and deletion of some of the most highly expressed genes had no effects on cell proliferation. in addition, many genes necessary to maintain normal cell fitness under these treatments did not have significantly altered expression levels. large-scale mutant phenotyping monitors changes in mutant fitness or other visible traits, and provides direct assessment of the requirements of genes under specific conditions. the availability of the open reading frame deletion mutant collections in the budding yeast s. cerevisiae and the fission yeast schizosaccharomyces pombe has proven to be advantageous in this approach. the principal challenge in large-scale phenotyping is distinguishing individual mutants. the current method of choice is to tag each mutation with a unique dna sequence called a “barcode”. because barcode tags are part of the mutations, the proportion of an individual barcode reflects the proportion of that barcode-associated mutant in a population. thus, following barcode frequencies can identify mutants with a desired growth phenotype from a population of diverse mutants, an approach referred to as parallel analysis. an advantage of parallel analysis of barcoded mutants is that mutations causing deleterious or weak phenotypes can be efficiently detected. for example, to identify virulence genes in salmonella typhimurium, mice were infected with a s. typhimurium mutant library where each mutant carried a unique bp barcode tag. the barcodes that were lost from the post-infection population identified the genes required for virulence. this approach has also been extended to cultured human cells using barcode-tagged cdna and shrna libraries to discover genes whose overexpression and down-regulation could suppress cancer cell growth and survival. in all three screens, the mutants of interest diminished in the population but could be revealed by detecting the loss of their associated barcodes by dna arrays. the budding yeast s. cerevisiae is the model organism where barcode-tagged mutagenesis has been the most successfully exploited. its small, sequenced and well-annotated genome and the efficient gene deletion techniques allowed the production of a collection of complete orf deletion mutants where each deletion mutant is tagged by two unique barcodes. the barcodes can be amplified en masse by pcr to generate probes for commercially available high-density microarrays to take a “census” of the relative abundance of each mutant in a culture under a variety of conditions. two independent genetic screens using this barcode-tagged deletion mutant set have identified many genes whose deletion caused lengthened chronological lifespan as detected by an increase in the abundance of the long-lived mutant barcodes. as long-lived mutants and mutants with normal lifespan are often morphologically indistinguishable, the barcode approach to monitor the length of lifespan of many mutants in parallel demonstrated the power of parallel analysis for detecting weak phenotypes. s. pombe is an important model system that has many of the advantages of s. cerevisiae, including a sequenced genome and well-established microbiological, genetic and molecular biological approaches. unlike s. cerevisiaes. pombe shares a number of similar features with mammals including rna interference, aspects of rna splicing and the requirement for the mitochondrial genome for survival of wild type cells. a barcode-tagged deletion strain set for s. pombe, in which each deletion mutant carries two barcodes, has also been developed recently. most of the mutants in the s. pombe and s. cerevisiae orf deletion sets are null mutations, and haploid mutants lacking essential genes are not present. truncated essential proteins with partial or altered function and mutated alleles of essential genes with reduced expression levels have been shown to produce viable and detectable phenotypes such as changes in transcriptional silencing and lifespan. therefore, a mutant collection generated by a different approach that includes mutations that impair the function of essential and non-essential genes would be beneficial for parallel analysis of phenotypes and genome-wide screens. an alternative mutagenesis method in s. pombe is non-homologous recombination-dependent integration of a selectable marker, which can generate a wide variety of mutations. insertions in the coding sequences can produce truncated proteins with either no or altered function. insertions in the 5’ and 3’ region of a gene can change protein expression levels by compromising promoter function and mrna stability, respectively, and produce viable mutations in essential genes. previous studies in s. pombe characterized two classes of transformants when cells were transformed with linear dna that had limited or no homology to the genome. one class of transformants contained single or tandem copies of linear dna stably inserted in the genome, and the insertion events were often accompanied by deletions in the integrated dna vectors and adjacent genome sequences. the distribution of insertions in the genome appeared random. the other class, which constituted the majority of the transformants, maintained the transfected dna as unstable, extrachromosomal circular dna despite the absence of a known origin of dna replication in the vector. chua et al. and davidson et al. used this approach to create random s. pombe mutants by transforming a ~ kb dna fragment containing the ura4+ gene into strains with the ura4-d mutation, a deletion that removes the sequences homologous to the kb ura4+ dna from the genome. these studies revealed two important features. first, only one insertion event was identified in each mutant by the criteria of southern blotting. thus, it is possible to generate a library of transformants where each individual contains a single mutation. second, many of the transformants contained insertions composed of multiple full or partial copies of the vector at each site. the complex structure of these insertions made mapping their genomic locations challenging. to facilitate genome-wide functional analysis in s. pombe, we generated a barcode-tagged s. pombe library of random insertion mutants that retains features equivalent to the budding and fission yeast orf deletion mutant collections, enables identification of mutants in the absence of knowing the barcode sequences, and allows parallel analysis with basic molecular biology techniques. we created an insertion dna cassette composed of the ura4+ selectable marker, a random barcode, a “buffer” sequence to protect the barcode from degradation during integration, and other sequences to allow modifications of the integrated vector. of the ~ insertion mutations generated, phenotypic analysis and mapping of a subset indicated that the insertions were broadly distributed in the genome. thus, this work demonstrates the feasibility and potential utility of constructing barcoded, random insertion libraries in s. pombe, and provides approaches that can allow rapid analysis of large collections of barcode-tagged mutations in other organisms.  a linear dna vector loses sequences from the ends during non-homologous recombination-mediated insertion in s. pombe as a first test of using insertion mutagenesis to generate barcode-tagged insertion mutations in s. pombe, an initial barcoded insertion vector was made by pcr amplification of the ura4+ gene using ura4+-specific primers to produce a ura4+ gene with a random barcode sequence at its 3’ end. cells with the ura4-d mutation were transformed with the linear vector dna and maintained on minimum medium without uracil to select for ura+ transformants. anticipating that the majority of the transformants would carry extrachromosomal ura4+ circles, the initial transformants were re-grown twice on non-selective medium to allow cells to lose unstable ura4+ circles. these colonies were then replica-plated onto both selective medium and minimum medium plates with 5-fluoroorotic acid to identify transformants with stably integrated ura4+, which are inviable in 5-foa medium. the initial test with ura+ transformants identified eight that were sensitive to 5-foa, indicating that only about 4% of the transformed cells contained stably integrated dna. the rest of the transformants likely carried the ura4+ marker extrachromosomally and had lost the circular ura4+ dna upon growth on 5-foa-containing medium. analysis of the eight ura4+ insertion mutants by pcr revealed that seven mutants had lost their barcodes, indicating that successful integration was frequently accompanied by deletion of barcodes. these results are consistent with previous observations of low frequency stable integration by non-homologous recombination and deletion on the ends of the inserted dna, underscoring the need to increase the proportion of stable transformants and to prevent deletion of barcodes. construction and characterization of the bacterial barcode-tagged insertion dna library the final insertion dna vector contains a barcode between a “buffer sequence” and the 5’ end of the ura4+ gene so deletions would first occur in the buffer sequence or the selectable marker before altering the barcode. this buffer sequence included new sequence elements with additional potential functions: a modified bp λ dna sequence at the 5’ end to prevent the degradation of the barcode, followed by a modified human hsp promoter that contains a lexa binding site, and a mutated loxp site named lox. the lexa-hsp promoter can be bound by any lexa fusion protein and may stimulate transcription through the λ dna sequences, which have been modified to contain no atg sequences, and would translate the first atg in s. pombe genomic dna. the lox site can, in the presence of cre recombinase, recombine with the lox site to allow integration of a plasmid. the new barcode consists of a 27-nucleotide random sequence that is interrupted at specific positions with a bases. this “interrupted barcode” is flanked by two sfi i restriction sites so that the barcodes can amplified by pcr, digested with sfi i restriction endonuclease and oligomerized in a head-to-tail orientation for sequencing several barcodes in a single reaction. as a test of this approach, a fragment containing the barcode dna and flanking insertion vector sequences was first pcr amplified and then digested with sfi i. gel-purified barcode monomers were then ligated together to form oligomers. a ladder of barcode oligomers was generated, and cloning and sequencing of those oligomers longer than five barcodes confirmed that they were ligated in a head-to-tail manner. we have successfully cloned and sequenced up to tandem barcodes per plasmid, and could routinely clone an average of seven barcodes per plasmid in pilot experiments. thus, when sampling a population of random mutants, each sequenced plasmid provides information on about seven different barcodes and sequencing to plasmids can provide a sufficiently large sample size to identify barcodes that are present at high frequency. as the barcodes used in this work were designed with random bases, they provide a total of or × possible barcode sequences. we generated a barcode library of × bacterial clones from sub-libraries containing × to × clones per sub-library. each sub-library contains independent and different random barcodes and helps preserve the high complexity of the library. our goal was to generate fission yeast mutants with unique and non-redundant barcodes, which should give a 76% chance to cover all annotated genes. in general, the number of fission yeast mutants produced was ≤ 1% of the total number of barcode clones present in the bacterial sub-libraries used for mutagenesis, so all mutants have a ≥ 95% chance of having unique barcodes. construction of the fission yeast barcode-tagged insertion mutant library the kb linear barcoded insertion vector dna was purified from the bacterial barcode sub-libraries after digestion with bamh i enzyme and used to generate the initial transformants. to enrich the overall proportion of stable integrants, we tested the possibility of utilizing low concentrations of 5-foa to select against cells bearing multiple copies of extrachromosomal ura4+ circles. cells bearing ura4+ circles are expected to have multiple ura4+ genes per cell. higher levels of ura protein should produce more toxic 5-fluorouracil in the presence of low concentrations of 5-foa, causing these cells to have a growth disadvantage. in contrast, stable integrants with one or few copies of integrated ura4+ dna do not produce as much ura protein, and are less sensitive to the same treatment. to test this assumption, fission yeast cells transformed with the linear insertion vector dna were immediately plated on low-dose 5-foa selective medium plates. the resulting transformants were verified for stable integration by growing them on normal-dose 5-foa medium and minimum medium plates without uracil. an initial test on transformants grown on low-dose 5-foa medium plates identified transformants which died when transferred to medium with normal-dose 5-foa plates. this result indicates 30% of the transformants contained stably inserted vector dna, a significant enrichment of mutants with stable integration compared to the previously observed 4%. the low-dose 5-foa selection procedure was used in large-scale generation of fission yeast insertion mutants. transformants obtained on low-dose 5-foa selective medium plates and passing the first verification on normal-dose 5-foa medium plates were transferred to non-selective medium in 96-well plates. each four 96-well plates were then used to make one 384-colony array on a selective medium and a normal-dose 5-foa omni plate to confirm the ura+ and 5-foa-sensitive phenotypes of stable insertion mutants. mutants behaving as true stable insertion mutants were stored individually in 384-well plates. in addition, five ~ -mutant pools and one ~ mutant pool were made for a total of ~ mutants. the insertion mutant library contains diverse mutations to evaluate the diversity of mutations in the insertion mutant library, four genetic screens were performed on mutants from ten 384-well plates for phenotypes that could be easily scored. in the first screen, we looked for mutants that grew slowly or could not grow on minimum medium plates supplemented with only adenine, leucine and histidine to complement the original auxotrophic mutations in our strain. we discovered mutants containing mutations that impeded growth enough to prevent visible colony formation on minimal medium. based on the gene ontology database -predicted mutant frequency and the fact that only ~57% of the s. pombe genome contains protein coding genes in which mutations are more likely to generate detectable phenotypes than in intergenic regions, auxotrophic mutants would be expected in the insertion mutants assayed. because not every mutation will generate a visible phenotype, the frequency of auxotrophic mutants in this assay was consistent with the go prediction. % a. the estimated number is from amigo database by searching genes using keywords “amino acid biosynthesis” and “nucleobase biosynthesis”, and excluding genes involved in the adenine, histidine, leucine and uracil pathways as the parental strain in defective in these pathways and all mutants are ura+. b. total gene number in s. pombe genome = c. the estimated number is calculated as “the total number of mutants tested ” × “amigo expectation” × “the fraction of protein coding sequences in the total s. pombe genome ”. d. not determinable; white colony color could also result from mitochondrial defects. e. the estimated number is from kegg. f. not determinable; no associated terms or categories in amigo or kegg. in the second assay, we selected for mutations that altered colony color on low adenine medium. as the parental yeast strain of the insertion library mutants contains the ade6-m mutation, we screened for an alteration in colony color from pale pink to red or white when cells were grown in rich medium with low concentrations of adenine. mutations in only two genes, ade6+ and ade7+, are known to cause red pigment accumulation in cells. one mutant out of the screened showed dark red colony color on low adenine medium plate and was identified as an ade7- insertion mutant. thirteen mutants which turned white on low adenine medium plates were also isolated. although this phenotype can be associated with mutations in the adenine biosynthesis pathway that produce substrates for the ade and ade enzymes, mutants with compromised mitochondrial function have also been reported to demonstrate a similar phenotype. in the third test, we screened for temperature-sensitive mutations that allow mutant cells to grow normally at 30°c, but not at 36°c. a total of such mutants that grew slowly or could not form visible colonies at 36°c were recovered. mutations that cause temperature-sensitive growth are not well-characterized, so these data cannot be used to estimate the expected mutant frequency. however, the isolation of temperature-sensitive mutants does demonstrate the wide variety of mutations present in the library. in the fourth experiment, we selected for mutations that confer cellular resistance to ethidium bromide. etbr toxicity is primarily due to inhibition of the circular mitochondrial genome. as with mammalian cells, s. pombe requires a functional mitochondrial genome for survival so etbr is highly toxic. if s. pombe and mammalian cells acquire certain nuclear mutations, the so-called rho cells devoid of mitochondrial dna can be generated by long term selection in etbr-containing medium with specific supplements. using this etbr selection procedure, we identified strains that are etbr-resistant. to determine whether these mutant phenotypes were caused by the ura4+ insertion or mutations induced by transformation, we crossed the mutants to a wild type strain and performed tetrad analysis on a subset of the identified mutants to verify co-segregation of the ura4+ marker and the phenotypes. of strains examined, showed 2: segregation of ura4+ in seven tetrads. by this criteria, the majority of mutants contained single insertions. of the mutants that showed slow or no growth on minimum medium, the ura4+ marker co-segregated with the phenotype in a 2: ratio in mutants, indicating that most of the mutant phenotypes were linked to the insertion mutations. similar results were observed in mutants exhibiting altered color on low adenine medium. of the temperature-sensitive mutant assayed, one was sterile, four showed poor spore viability and three of the remaining six showed 2: co-segregation with ura4+. the source of the unlinked mutations is unknown but may be due to the mutagenic effects of transformation, and underscores the requirement of validating mutants from premade collections by recreating the mutation in a new strain to determine if the phenotype is regenerated. the sum of these data show that the majority of these insertions contain the ura4+ vector in a single locus and the insertion locus is genetically linked to the mutation in most of these mutants. mapping of insertion sites and analysis of the structures of inserted vectors previous studies on non-homologous recombination-mediated mutagenesis in s. pombe showed that the genomic locations of insertion vectors were difficult to determine by inverse pcr. we therefore tested two previously established methods for large-scale insertion site determination, thermal asymmetric interlaced -pcr and splinkerette-adaptor pcr. tail-pcr uses alternating high and low annealing temperatures, a set of arbitrary degenerate primers and three nested insertion dna-specific primers to amplify a small region of insertion dna and the adjacent genomic sequence. tail-pcr only detected genome-insertion junctions in a portion of mutants analyzed. in some mutants, only repetitive insertion vector sequences were amplified, indicative of tandem integration of vector dna in these mutants. in other mutants, mitochondrial dna was found co-integrated with insertion vector. these results indicate that these additional dna fragments provided binding sequences for the degenerate or vector-specific primers. therefore, we also used splinkerette pcr, which involves ligating a double strand dna adaptor to genomic dna fragments digested with restriction enzymes that cut frequently in the genome but not in the vector. the resulting products provide templates for pcr amplification of neighboring genomic dna using specific primers on the splinkerette adaptor and insertion vector. the splinkerette approach produced pcr fragments of defined size and some genomic sequences, allowing the estimation of the region of the insertion in some mutants and detection of insertion-chromosome junction in others. as the splinkerette approach also gave vector or mitochondrial sequences in some mutants, we pursued additional mapping approaches. we adapted splinkerette and inverse pcr to a new method, inverse splinkerette pcr, which may eliminate extra copies of tandem insertion vector in some mutants before pcr. in inverse splinkerette pcr, genomic dna from insertion mutants was digested with a restriction enzyme that cuts once in the insertion vector and frequently in the genome. the digestion generates fragments with one chromosomal dna end and one insertion vector end, which can be brought together by intramolecular ligation to produce circular dna. digestion of the ligated dna with sfi i enzyme linearizes circular dna composed of truncated 5’ part of the insertion vector and a genomic fragment, and generates a unique overhang on the λ buffer of vector for ligating splinkerette adaptor. the genomic dna bordered by the partial vector dna and splinkerette adaptor can be amplified by specific primers on insertion vector and splinkerette. this method can reveal the ecor v site closest to the insertion site and together with the length of the pcr product, to generate an approximate location of the insertion mutation. in cases where the ecor v site is very close to the insertion site, one may detect the precise junction sequences between chromosomes and insertion vector. we also directly cloned the genomic sequences flanking the insertion vector in e. coli by the lox66/lox integration system. as the insertion mutations are all marked with the mutated loxp sequence, lox introducing a plasmid bearing the lox sequence into cells expressing cre recombinase should allow stable integration of the lox plasmid into the lox sequence. we therefore transformed the plasmid plox which has the lox sequence and a selectable marker for g resistance in yeast and kanamycin resistance in e. coli, into s. pombe insertion mutant cells bearing the prep plasmid with or without the cre recombinase gene. because plox does not contain a functional yeast replication origin, integration into the genome is required for its stable inheritance and g resistance in yeast cells. following introduction of plox cells expressing cre recombinase produced more g418-resistant colonies than cells without cre, indicating that cre promotes efficient integration of plox into the genome. specific integration of plox to lox of the insertion vector was verified by pcr using primers on plox and the insertion vector. while expected pcr products were generated from cre-expressing cells, the same pcr reactions using those g418-resistant cells without cre did not yield bands corresponding to plox integration, indicating these cells may bear plox extrachromosomally. sequencing of pcr products from cells expressing cre also confirmed that they contained the recombined lox66/lox hybrid and the wild type loxp sequences. to clone genomic dna flanking the insertion sites, genomic dna of g418-resistant, plox66-integrated cells was digested with spe i and xba i, and ligated under conditions favoring intramolecular ligation. the presence of the puc origin on plox allowed us to clone genomic sequences neighboring the insertion site in e. coli for sequencing. using this method, we cloned and verified the insertion site of the mutant 18_m. using tail-pcr, we initially characterized mutants, of which we were able to determine the genomic sequences at one end of the insertion sites in mutants. in mutants, only repetitive insertion vector sequences were amplified and sequenced, and mitochondrial dna was found co-integrated with insertion vector in eight mutants. we have also used inverse splinkerette pcr to map insertions in mutants and determined approximate location of the insertion vector in mutants, nine of which we were not able to determine by tail-pcr. these results suggest that although tail-pcr and inverse splinkerette pcr are not sufficient for mapping all insertion sites, they are complimentary to each other in deciphering insertion positions accompanied by different insertion structures. splinkerette pcr and lox66/71-based cloning also allowed the determination of insertion sites. among the mutants in which exact or approximate insertion locations were determined, of them have insertion mutation on chromosome with on chromosome and on chromosome in the mutants where at least one end was mapped to a defined locus, were located in orfs or non-coding rna genes and were found in intergenic regions. in the seven mutants in which both ends of the insertion sites were characterized, deletions of genomic sequences were found in only three mutants, and deletions of a wide range of sizes, from bp to kb, were observed at ends of the insertion vectors in all mutants. tandem integration of multiple insertion vectors were detected in all but one mutant in head-to-tail, head-to-head or tail-to-tail orientations. the structures of the insertion vectors in these mutants is consistent with the ends of some linear vector dna being resected by nucleolytic activities in cells and ligated together before being integrated as tandem copies into the genome as previously observed by others. discussion in this study, we created a fission yeast insertion mutant library in which all mutants were tagged with unique barcode sequences and stored as two readily available selection platforms. the 384-well mutant arrays allow genetic screens on individual mutants and can be extended to genetic approaches such as synthetic genetic array. these mutant arrays have been used to identify mutants with four distinct phenotypes as well as strains that are hyper-sensitive to cancer chemotherapeutics camptothecin and bleomycin. in addition to 384-well mutant arrays, mutant pools of mutants are available for parallel analysis. the insertion mutagenesis used in this study relied on random non-homologous recombination, where a vast majority of transformants have unstable, circularized vector dna and only a small portion have stable insertions. to facilitate the collection of stable insertion mutants, we included low-dose 5-foa in our initial selecting medium as an effort to eliminate unstable cells bearing high copy number of ura4+ vector and producing high levels of ura4p, and subsequently re-screened for mutants that were stably ura4+ and 5-foa-sensitive. while this approach increased the proportion of stable insertion mutants among the total transformants, we note that some mutants might have been excluded. for example, insertions into genomic regions where expression switches between on and off states would be excluded from the final library. likewise, insertion at a locus that causes high ura4+ expression or tandem integration of many functional ura4+ markers could result in increased sensitivity to low-dose 5-foa and eliminate some mutants during the initial selection. to increase the versatility of the mutant library, some previously characterized functional dna sequences were included in our insertion vector, including the lox sequence and the mutated human hsp promoter. we demonstrated that the mutated lox and lox could undergo cre recombinase-dependent integration in s. pombe, similar to what has been reported in mammalian cells, and showed that this method can be used to clone genomic sequences surrounding the insertion mutations using the plox plasmid. the mutated human hsp promoter, which exhibits dramatically reduced activity in s. pombe, was tethered with a lexa binding site for its potential activation by a lexa dna-binding domain and transactivator fusion protein. while the goal was to provide an opportunity to ectopically express nearby genes, the tandem integrations of insertion vector and co-integration of mitochondrial dna could impede the utilization of this promoter to activate genes near the insertion site. testing of the hsp promoter was, therefore, not pursued. another novel addition to this insertion mutant library is the inclusion of unique barcodes. these barcodes allow one to take a census of the selected mutants by tracking barcode frequencies after the selection. barcode sequencing can be facilitated by converting individual sfi i site-bordered barcodes to barcode oligomers, which allows for the generation of multiple barcode sequences per sanger sequencing reaction, providing a cost-effective and rapid alternative compared to cloning and sequencing individual barcodes. although the high-throughput sequencing or microarray is possible analytic means for our mutant library, the design of our barcodes provides a medium-throughput alternative which requires only easily accessible laboratory techniques. moreover, in contrast to the necessity of sophisticated bioinformatics support for microarray and high-throughput sequencing, the sequences of several hundred barcodes obtained from our approach can be easily sorted and analyzed with basic spreadsheet software. we note that our oligomerization and sequencing strategy can also be used to monitor other nucleic acids in cells. by amplifying a small region around a sequence difference between two different nucleic acids, our oligomerization and sequencing method can easily produce ~ or more sequences, which should be sufficient for determining the relative proportions of the two forms with data that are much easier to process than data from a high-throughput sequencing experiment. random insertion mutant libraries may overcome some challenges in the study of essential genes. mutants that lack essential genes are not present in haploid gene deletion mutant banks such as the budding and fission yeast orf deletion collections. among the s. pombe insertion mutants analyzed in this work, we found that in six mutants, the insertions were identified in or adjacent to essential genes, presumably generating truncated proteins or altering the expression of these genes. these results indicate that this insertion mutant library approach provides opportunities for functional analysis of essential genes. the insertion events characterized in this library are consistent with those shown in previous studies, including insertions in both genes and intergenic regions, large deletions of insertion dna and little or no deletions of surrounding chromosomal sequences. we also discovered mutants with mitochondrial dna co-integrated with the insertion vector. the presence of mitochondrial dna in the wild type s. pombe nuclear genome has recently been characterized with one wild type strain containing mitochondrial dna insertions in its nuclear chromosomes. mitochondrial dna fragments were also found in all repaired plasmid-based double strand breaks in s. pombe cells in an independent study. it is worth noting that capture of mitochondrial dna in the nuclear genome has also been observed in hemiascomycetous yeasts, plant, insect, rodent and human cells and appears to be an active and ongoing process, indicating that s. pombe transformation provides a way to study this process. one consequence of mitochondrial and tandem ura4+ dna insertions is that they can impede the detection of the insertion sites by tail-pcr. we have tested three additional approaches for mapping insertion mutations: splinkerette pcr, inverse splinkerette pcr and lox66/71-dependent cloning. while these methods were not 100% efficient, we showed that they could complement each other in defining insertion sites. one advantage of tail-pcr is that it detects the junction of insertion vector and genomic sequences and provides the exact location of the integrated vector. in contrast to tail-pcr, inverse splinkerette pcr in our mutants directly determined the closest ecor v sites to the 5’ end of insertion vector. together with the length of the pcr products, only approximate regions of insertion could be obtained. while we did not follow up results from our inverse splinkerette pcr, one could determine the exact location of insertion by cloning the pcr product and sequencing the genomic regions with gene-specific primers. depending on the complexity of insertion structure, splinkerette pcr could determine the insertion vector-chromosome junction or the closest restriction sites used in the assay to the insertion site. it is important to note that all mutants are tagged by the lox sequence, which allows the cloning of the genomic sequences flanking the insertion in e. coli in the event that insertion mutations could not be mapped by these three pcr methods. in addition to non-homologous recombination-based integration, other methods for generating insertion mutations include transposon-mediated mutagenesis. at least three types of transposons have been analyzed in a genome-wide context in s. pombe. the s. pombe retrotransposon tf has been shown to exhibit preference for targeting the promoters of rna polymerase ii transcribed genes. the piggybac transposon, originally isolated from cabbage looper moth, preferably targets ttaa sites in the genome. although as much as 79% of transposition events of piggybac analyzed in a haploid s. pombe strain was located in intergenic regions, it was assumed that this seemingly preference of pb transposon for intergenic sequences was a consequence of selective pressure on insertions in orfs that cause reduced fitness. high throughput sequencing performed in both studies indicated that the transposition events of both transposons broadly distribute among the three chromosomes. the hermes transposon from housefly musca domestica has strong preference for t at position and for a at position of the target sites and has been adapted for s. pombe. the limited number of insertion mutants analyzed in that study suggested that hermes targets both intergenic regions and coding sequences with no apparent bias. insertions generated by non-homologous recombination in our work also have a broad distribution on the three chromosomes, similar to what was observed in tf retrotransposon and pb transposon and a previous report on non-homologous recombination in s. pombe. although 60% of the partially characterized insertions resided in orfs or non-coding rna genes, the enrichment of this type of insertions may be due to pre-selection of the corresponding mutants by visible phenotypes. thus, the three transposon approaches and our insertion vector approach can create a wide variety of mutations. our approach has the advantage of adding unique barcodes to each insertion, which has not been applied to the transposon approaches. the main difference between non-homologous integration and transposon transposition is the structures of insertion events. while transposons generally integrate at individual genomic locations as unmodified single copies with defined junctions between genomic and transposon dna, tandem integration of the insertion vector dna or co-insertion of non-nuclear dna during non-homologous recombination-based integration make this junction more variable. the simple insertion events in transposon mutagenesis allow for high-throughput sequencing for mapping insertion sites, while the complex insertion structures generated by non-homologous recombination require insertion sites to be determined by low/medium-throughput approaches. nonetheless, the high mutation variety, the presence of random barcodes, and the availability of multiple methods for mapping insertion mutations still make this insertion mutant library an attractive tool for genome-wide studies that can complement the existing s. pombe orf deletion set.  as an effort to further support genome-wide studies in s. pombe, we generated a barcode-tagged s. pombe insertion mutant library which is available as pools of mixed mutants for parallel analysis and in the form of 384-well mutant arrays for genetic screens on individual mutants. the four genetic screens conducted in this work indicate that the library has a wide variety of mutations and is suited for the selection for diverse phenotypes. the design of the barcode tags allows detection and quantification of the barcodes with readily available molecular biological techniques, and does not require prior knowledge of the barcodes and mutations in the mutant strains to conduct genetic screens. the barcode-tagging strategy described here can be easily adapted to other model systems. 
an insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. we present several approaches to determine insertion sites. individual barcodes are flanked by sfi i recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. while these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. this collection of s. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. to further support genome-scale research in s. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. barcodes are unique dna sequence tags that can be used to specifically label individual mutants. the barcode-tagged open reading frame haploid deletion mutant collections in the budding yeast saccharomyces cerevisiae and the fission yeast schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes.
 barcodes are unique dna sequence tags that can be used to specifically label individual mutants. the barcode-tagged open reading frame haploid deletion mutant collections in the budding yeast saccharomyces cerevisiae and the fission yeast schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. while these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. to further support genome-scale research in s. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques.  an insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. individual barcodes are flanked by sfi i recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. we present several approaches to determine insertion sites.  this collection of s. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. the design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. 