BACKGROUND
microbes are critical symbiotes for humans, where upwards of  <dig> trillion foreign cells from more than  <dig> different species reside  <cit> .
the gut is host to the bulk of the microflora, where bacteria are the most abundant, outnumbering eukaryotes and viruses by orders of magnitude.
while a handful are known human pathogens, the majority of these bacteria, such as lactobacillus sp.
are commensal or mutualistic, exerting their influence through probiotic functions  <cit> .
studies in mice and humans implicate gut bacterial influence not just in digestion of nutrients  <cit> , but in fat storage  <cit> , modulation of bone-mass density  <cit> , angiogenesis  <cit> , protection against pathogens  <cit> , and immune functions  <cit> .
conditions such as crohn’s disease  <cit> , diabetes  <cit> , and obesity  <cit>  have all been directly linked to an imbalance of gut microflora.
despite an explosion of research in recent years, the ecology and mechanistic details of complex microbiomes such as those found in the gut remain enigmatic, and new methodologies for dissection and characterization are needed.
metagenomics refers to a powerful set of genomic and bioinformatic tools used to study the diversity, function, and physiology of complex microbial populations  <cit> .
substantial advances in microbiome research have been driven by the extensive use of next generation sequencing  technologies, which allow annotation and characterization of microbiomes using targeted  or shotgun approaches  <cit> .
targeted approaches are suboptimal in the identification of low abundant species  <cit> , and even though identification of most species from a population is possible using shotgun sequencing, assembly of complete genomes of individual species is rarely possible unless those species are highly abundant.
moreover, as complexity increases, dataset resolution decreases, reducing the ability to comprehensively analyze community structure.
recent reports provide promising advances in metagenomic binning and assembly for the reconstruction of complete or near-complete genomes of rare  community members from metagenomes.
albertesen et al.
<cit>  have described differential-coverage binning as a method for providing sample-specific genome catalogs, while wrighton et al.
<cit>  have also been successful in sequencing more than 90% of the species in microbial communities.
in another approach, either gc content  <cit>  or tetranucleotide frequency  <cit>  combined with genome coverage patterns across different sample preparations was used to bin sequences into separate populations, which were then assembled under the assumption that nucleotide  frequencies are constant for any specific genome.
sequencing throughput is continually improving and is expected to provide access to increasingly lower abundance populations and improvements in read length and quality will reduce the impact of co-assembly of closely related strains  on the initial de novo assembly.
while these approaches represent exciting advances in bioinformatic tools, experimental tools for reducing the complexity of a population prior to sequencing, such as enriching for low abundant organisms or intact cells, provide alternative and complementary approaches to improve genomic analysis of such complex systems  <cit> .
a variety of experimental methods have been used to decrease sample complexity prior to sequencing.
the most commonly used tool for decreasing sample complexity is probably single cell genomics   <cit>  which utilizes flow cytometry, microfluidics, or micromanipulation to isolate single cells as templates for whole genome amplification by multiple displacement amplification   <cit> .
as it requires only a single template genome, it allows the sequencing of “uncultivable” organisms.
for example, a recent paper from the quake group used microfluidics to isolate single bacterial cells from a complex microbial community, using morphology as discriminant, before genome amplification and analysis  <cit> .
scg approaches rely on mda, and while mda can generate micrograms of genomic amplicons for sequencing from a single cell, amplification bias, leading to incomplete genome coverage, is a major inherent limitation  <cit> .
in fact, a recent survey of  <dig> genomes sequenced from single cells had a mean coverage of approximately 40%  <cit> .
a clever use of single amplified genome  assembly improved coverage to >90% for  <dig> of the  <dig> genomes, with mean coverage being approximately 70% for the  <dig> genomes when assembled from multiple sags.
mda-associated amplification bias has been improved for eukaryotic cells using a technique called malbac  <cit> , but these improvements have yet to be shown for prokaryotic genomes and still rely on random, or morphologically based, cell sorting.
such random sorting of single microbial cells from complex mixtures is expected to bias against rare species and may require sorting and sequencing of hundreds to thousands of cells before a rare genome can be obtained.
increased input template number can overcome mda amplification bias, or difficulties in processing and sorting single cells from biofilms, and provide near complete genome coverage.
potential methods for accomplishing this include inducing artificial polyploidy or using gel microdroplets  <cit> .
however, in both of these cases, rare species may still be missed if sufficient numbers of single cells cannot be sorted.
this has been partially addressed in a recently published “mini-metagenomics” approach.
mda product coverage was improved by creating bacterial pools by flow cytometry, with ~ <dig> bacteria in each pool.
screening of these pools for 16s rdna sequences of the bacterial species of interest, followed by deep sequencing of the positive pools, allowed assembly of a relatively complete genome from different pools containing the same 16s rna sequences  <cit> .
an alternative approach to simultaneously address both amplification bias and isolate rare species is to use antibodies recognizing specific microorganisms within microbial communities to enrich and/or subtract bacterial species prior to sequencing.
we hypothesized that enrichment by selective sorting in this way could provide a powerful method for significantly increasing input template number to obtain complete genomes of low abundance species, akin to creating a small microbiome in which all members expressed a single target recognized by the antibody of interest.
in the present work, we developed a selection and screening pipeline using phage display and flow cytometry to isolate a single chain fv  antibody that can: i) identify a bacterial species, lactobacillus acidophilus, with extreme specificity; and ii) be applied to a microbiome, using fluorescence activated cell sorting , to identify, enrich, and deplete targeted species from bacterial mixtures.
we further demonstrated that if this approach was applied to a mock community containing l. acidophilus, rather than the pure single species, antibodies recognizing l. acidophilus could be isolated.
this phage display selection method is highly adaptable to recognition of any organism and provides a unique tool for dissection and sequencing of rare species from complex microbiomes.
RESULTS
selection against intact bacteria using phage display and screening by flow cytometry
we chose the probiotic lactobacillus acidophilus atcc  <dig> as a target for our approach.
lactobacilli such as sp.
acidophilus are widely studied gut microbes with probiotic functions including digestion, immune function, and prevention of diarrhea  <cit> .
antibody selections were performed against l. acidophilus using two methods.
in the first, the bacteria were coated on immunotubes , while, in the second, selection was carried out by centrifugation.
for each selection we used a previously described naïve scfv library displayed on m <dig> filamentous phage  <cit> .
two to three rounds of selection, with increasing stringency, were performed prior to re-cloning enriched scfvs into pep-gfp <dig>  <cit>  for screening.
this vector generates scfv proteins in fusion with two different detection tags: sv <dig>  recognized by a monoclonal antibody  <cit>  and s <dig>  a split green fluorescent protein  tag that fluoresces when complemented with gfp1- <dig>  <cit> .
the simultaneous use of both tags enhances signal-to-noise ratio when testing putative clones for binding activity against l. acidophilus in flow cytometry.
scfv culture supernatant was incubated with l. acidophilus followed by staining and the l. acidophilus bacteria analyzed using an lsrii flow cytometer .
sequencing revealed one unique scfv  from the immunotube selection, and three unique scfvs  from the selection by centrifugation .
the α-la <dig> scfv was found to be highly specific for l. acidophilus, binding to all tested l. acidophilus strains , but not to a panel of other gut bacteria, including bifidobacterium sp., peptoniphilus sp., e. coli, and six different species of lactobacillus .
our analysis included lactobacillus helveticus, the closest species to l. acidophilus, the 16s rrna sequence of which shares >98% identity  <cit> .
the other three α-la scfvs showed similar degrees of specificity.
we proceeded with the α-la <dig> scfv for the remainder of the study due to greater expression and apparent affinity relative to the other α-la scfvs .
the specificity of the α-la <dig> scfv was also further validated using the amnis image-stream mark ii flow cytometer , which captures microscope images in a flow cytometric configuration .
lactobacillus strains were grown in atcc no.
<dig> lactobacilli mrs broth.
all other strains were grown in atcc no.
<dig> reinforced clostridial broth with the exception of ec which was grown in luria broth.
the specific surface antigen recognized by all the α-la scfvs was identified as the l. acidophilus s-layer a protein,  using western blotting and mass spectrometry .
slpa proteins are highly abundant, paracrystalline surface glycoproteins that make obvious targets for scfv recognition  <cit> .
further analysis following deglycosylation of the bacterium revealed that recognition was not mediated by glycosylation of the protein .
scfv specificity to l. acidophilus in a mock community
we tested the use of the isolated α-la <dig> scfv protein to detect varying abundances of l. acidophilus within a mixture of different bacterial species.
we individually grew a total of ten species in their respective growth media .
the various species were mixed to generate a “mock” community, which enabled us to control the relative composition of different species within the mixture.
all species in the mock community were added at equal concentrations .
the four resultant mock communities contained 10% of each of these species, and differed only in their relative abundance of l. acidophilus at 10%, 5%, 1%, and  <dig> % in the community.
staining with purified α-la scfv was followed by analysis by flow cytometry.
pure l. acidophilus stained with α-la <dig> scfv was used to establish the l. acidophilus analysis gate  as reference for varied l. acidophilus abundances in the mock communities.
ten thousand events from each mock community were analyzed.
we observed  <dig> %,  <dig> %,  <dig> %, and  <dig> % l. acidophilus in the mock 10%, 5%, 1%, and  <dig> % communities, respectively.
this degree of accuracy supports the possibility that the scfv can detect target bacteria within a population, with abundance less than  <dig> %, and further supports the specific nature of the α-la <dig> scfv.
targeted enrichment of single l. acidophilus cells from yogurt microbial community
the ability to sort single l. acidophilus cells using the α-la <dig> scfv was subsequently tested on cultured yogurt, a natural, heterologous community the constituents of which are reported to include streptococcus thermophilus, lactobacillus delbrueckii subsp.
bulgaricus, lactobacillus delbrueckii subsp.
lactis, lactobacillus acidophilus, and bifidobacterium lactis.
our aim was to validate specificity and test the ability of our selected scfv to recognize l. acidophilus from a culture even though the scfv was selected against bacteria grown in the laboratory.
bacteria were isolated using methods previously described based on a series of density gradient centrifugations to remove sample debris prior to bacterial cell isolation  <cit> .
after staining with α-la <dig> scfv-gfp + α-sv5-pe ,  <dig> -5% of the total population, depending upon the yogurt preparation, fell into the l. acidophilus-specific gate  .
single bacterial cells were sorted from the pre-sort , negatively sorted , and positively sorted  gates for amplification by mda and subsequent 16s rdna sequencing.
we identified the species origin of  <dig> individual cells sorted from four different replicates .
the dominant species in the community was streptococcus thermophillus, with lactobacillus delbruekii and at least eight other species identified, including species that were not expected to be found in the yogurt culture.
on average, sequencing showed l. acidophilus recovery at  <dig> %  in the pre-sort  community, enrichment at  <dig> %  in p <dig>  and complete absence in p <dig> , thereby demonstrating the feasibility of species depletion.
in three of the replicates, l. acidophilus sequence was not observed in the pre-sort  sample , but was nevertheless enriched and identified in the p <dig> gate, indicating that the l. acidophilus likely would not have been identified using standard single cell sorting and analysis.
obtaining a complete genome using scfv targeted enrichment
one of the primary goals of this study was to show that targeted enrichment of template using phage derived antibodies and facs can be used to generate complete genome sequences of rare species, with the specificity conferred by the selected scfv enabling the enrichment of enough template to complete a genome without any further downstream cultivation or chemical treatment prior to mda.
to test this idea, l. acidophilus was sorted from one of the bacterial yogurt extractions,  as either single cell or 50-cell templates for mda, and sequenced using the illumina miseq platform.
for reference mapping, reads from both the single and 50-cell sorted amplicons were normalized and mapped to l. acidophilus ncfm .
in parallel, as reference genomes are unavailable in most cases, we also assembled the genome de novo using the normalized reads.
the assembly tool clc was used to both map reads and assemble contigs de novo.
having a reference genome available allowed us to accurately assess the extent of genome coverage using both mapped reads and de novo assembly.
as we hypothesized, reads mapping from the 50-cell template yielded near-complete genome coverage at  <dig> %, while the single cell template fell short at 68% with far more amplification bias .
bias is clear  in the single cell template with a large portion of the genome lacking coverage while other regions are covered at very high frequencies of > <dig>  fold.
for the de novo assembled genome, the 50-cell template yielded  <dig> contigs  with > <dig> % coverage of the reference and ~8-10% contamination by sequences from non-l. acidophilus species.
the contaminating non-lactobacillus reads were identified by searching assembled contigs in sequenced microbial genomes.
we found that the single cell data was contaminated with sequences from bacteria close to a sequenced pseudomonas genome  and the 50-cell data was contaminated with genomic sequences related to rhodopseudomonas , bradyrhizobium  and nitrobacter .
<dig> % of the single cell read data mapped to the pseudomonas genome and  <dig> % of the 50-cell data mapped to the rhodopseudomonas genome,  <dig> % to the bradyrhizobium and  <dig> % to the nitrobacter.
the contaminations were likely generated during the cell sorting and/or the mda process.
mda-related contaminants, such as non-specific amplification and dna presented in reagents, are common to virtually any approach that utilizes whole genome amplification  <cit> .
beside possible contamination from the mda process, most contaminants were probably introduced during the cell sorting process since contaminated sequences were not shared between single and 50-cell results.
we hypothesize that sorted specific cells may contain contaminating cells in the same droplet , or that contaminating dna, either free in solution or attached to the targeted cell may be sorted and become an mda template.
we believe it more likely that the rhodopseudomonas genome, which was 34% covered, may have been introduced by cell contamination, while lower level contamination may have occurred via the second mechanism.
fortunately, the vast majority of contaminant reads was easily removed and did not interfere with full data analysis of assembled contigs.
to assess coverage, de novo assembled contigs were mapped back to the reference and the resulting coverage was > <dig> % for the 50-cell template and 63% for the single cell.
these values are highly similar to those expected from draft coverage of cultured bacteria, indicating that template number enrichment using specific scfvs and facs can be used to sequence very low abundance  genomes in a community once a specific antibody is available.
selecting antibodies against a mock community
to determine whether this method can be applied to more complex microbial communities, we selected phage antibodies against the mock community used above, with each bacterial species present at ~10%.
selection was carried out by centrifugation, and after two rounds, the heavy chain complementarity determining region  <dig>  of the complete antibody output was sequenced by ion torrent.
the hcdr <dig> is the most diverse cdr, contributes most to antibody binding specificity, and is widely used as a surrogate for vh and scfv identity  <cit> .
using the antibody mining toolbox  <cit> , the hcdr3s of the antibodies selected against the mock community were identified and ranked for abundance.
as shown in table  <dig>  three of the twenty most abundant antibodies had hcdr3s that were identical to three of the previously selected antibodies  recognizing l. acidophlius, indicating that, in principle, it may be possible to select species specific antibodies directly against individual bacteria in complex bacterial communities, without the need to culture the individual bacteria.
however, validation of this possibility will require additional experimentation and selection on natural microbiomes rather than the mock community used here.
l. acidophilus
*total number of quality reads from the ion torrent sequencing run =  <dig> , <dig>
discussion
the expanding field of metagenomics continues to search for robust ways to obtain high-quality genomes from under-represented or rare species in a given sample.
improvements in sequencing throughput will enable access to lower abundance populations, but a “pre-enrichment/pre-clearing” step before the analysis can provide complementary and significant results.
we describe a novel and adaptable approach for sequencing low abundance genomes from microbial communities, with potential improvements in the genomic coverage of low abundance species where standard single cell approaches result in incomplete genomes or may have missed the organism altogether.
we demonstrate the use of phage display to select antibodies against a bacterial species with exquisite specificity.
the use of in vitro display potentially allows the method to be adapted to any organism or microbiome, does not rely on commercially available antibodies, and generates antibodies that are highly renewable and amenable to further engineering to modify affinity or specificity  <cit> .
to demonstrate the feasibility of the approach, we first targeted lactobacillus acidophilus, a bacteria naturally found in environmental samples from food to feces and is a principal commensal bacterium of the human gut.
the tested α-la <dig> scfv proved to be extremely specific and did not recognize other common gut microflora .
while it is practically impossible to prove that this scfv does not recognize any other bacteria, when tested on other lactobacilli such as l. helveticus, which is highly similar to l. acidophilus <cit> , we did not observe binding, providing strong evidence that the scfv is species-specific.
the target protein recognized by our scfv was identified as the surface layer protein a .
s-layer proteins are highly abundant and ubiquitous crystalline surface structures  <cit>  that have been implicated as a principal component for the organism’s probiotic functions  <cit> .
other lactobacilli tested in this study produce s-layer proteins that are highly similar  , but which can nevertheless be distinguished by our α-la <dig> scfv, demonstrating the high degree of specificity achievable.
since s-layer proteins are common to many bacteria, future work may involve re-engineering the α-la <dig> scfv to target s-layer proteins from other organisms, an option that is only possible with in vitro derived antibodies  <cit> .
coupling the specificity of phage-selected α-la <dig> scfv with facs allowed precise manipulation of a population on a per-cell basis, making possible the sufficient enrichment of l. acidophilus for > <dig> % genome coverage using both reference mapping and de novo assembly.
while it is common to observe this level of coverage for de novo assembly when the target organism is cultured prior to sequencing in the laboratory, the level of coverage reported here for a bacteria extracted from an environmental sample is exceptional.
for sequencing, we easily and rapidly sorted 50 l. acidophilus cells from an environmental sample  where l. acidophilus comprised ~ <dig> % of the population and were able to rapidly detect and quantify l. acidophilus at ~ <dig> % in a mock community comprising nine other species.
although we only tested compositions as low as ~ <dig> %, we are confident that l. acidophilus could be identified from mixtures where it is even lower in relative abundance with detection limited solely by the total number of cells available in a mixture and time available for sorting.
while detection and enrichment of rare species is an obvious use of these antibodies, depletion of common species may be equally important, as bias towards high abundance species is a well-known issue when performing shotgun metagenomics  <cit>  and, potentially, non-targeted single cell genomics.
our single cell analysis shows that l. acidophilus is completely depleted from the sample in the negative sort gate , demonstrating the feasibility of both depletion and enrichment.
separation methods, namely immunoprecipitation, micromanipulation, and flow cytometry have been described to improve genome sequencing, and the approach described here may also be applicable to other microbes found in microbiomes without being limited to organisms with innate fluorescence  <cit> , distinct morphology and/or high genome copy number  <cit> .
in this study we generated a scfv against an organism that can be cultured in the lab as a demonstration that recombinant antibodies can be raised against a specific organism and used to dissect, phylotype, and recover complete genomes for organisms from microbial communities.
we used an organism with a reference genome in order to accurately assess genome coverage.
future studies will involve selecting antibodies directly against uncultivable organisms within complex microbiomes.
we provide proof of principle, using selection against a mock community, that such an approach is potentially feasible: hcdr <dig> sequences of three of the antibodies selected against the pure culture were identical to those of antibodies selected against the mock community.
while this is promising, it is likely that selection procedures will have to be modified in order to select antibodies against the many different species present in a natural microbial community.
in particular, we have previously shown that selection against a specific antigen is far more efficient when carried out against the individual antigen than when the antigen is present in a mixture of other antigens  <cit> .
the situation is likely to be even more challenging for microbial communities, and may require selection in emulsions  <cit> , microfluidics  <cit>  or against individual cells  <cit>  to ensure that individual bacteria are isolated from one another during the selection process.
if the identity of the recognized bacteria in the microbiome is unimportant – i.e.
the goal is to catalog genome sequences present in a microbiome, whatever they are – the use of this method may be relatively straightforward.
it is likely to be more challenging, however, if the goal is to select antibodies against particular species in a population, unless an alternative means of bacterial isolation, such as fluorescent in situ hybridization  <cit> , is available.
one possible approach, which may be successful in microbiomes comprising few species, would be to select a panel of positive antibodies against different species within the community, and then deconvolute species recognition using facs and deep sequencing in a manner similar to that described here, after antibody selection and sorting.
however, the number of bacteria that can be extracted from environmental samples easily exceeds the number required for phage selection suggesting that this approach will be difficult for more complex populations.
since depletion is as feasible as enrichment using these scfvs with facs, it may be possible to iterate the process using scfvs against high abundance species for their subtraction and, thus, enrich for the low abundance organisms.
even if antibodies cannot be raised to low abundance organisms, depletion of high abundance organisms in a mixture will concentrate the low abundance ones, and so lead to improved taxonomic identification and genome recovery.
the described approach also has potential not only for the genome sequencing of novel and uncultivable organisms, but also in comparative genomics.
in this regard, selection of antibodies against organisms initially grown in the lab then used on environmental and clinical samples holds great potential for medicine and epidemiology  <cit> .
for example, a recent study  <cit>  reports the use of a commercially available igg antibody for targeted enrichment using immunomagnetic separation  to fully sequence chlamydia trachomatis directly from clinical isolates without culture.
our approach could extend on this work by adding a mechanism for the initial selection of suitable antibodies for studying pathogenic, probiotic, or other organisms.
near complete coverage, such as that provided by enrichment with phage-selected scfvs, is paramount for high resolution genomic comparisons.
in fact, while a discussion of genome differences is outside the scope of this study, we observed at least  <dig> single nucleotide polymophisms  when comparing the extracted l. acidophilus to the reference genome showing that the α-la scfv reported here could be used immediately for future comparative genome studies on human-derived l. acidophilus for both research and clinical purposes.
CONCLUSIONS
in this paper we demonstrate the power of combining phage antibody selection directly on bacteria with fluorescence activated cell sorting and deep sequencing to either enrich, or deplete, bacteria recognized by specific selected antibodies.
using this approach it becomes possible to assemble genomes directly from complex microbiomes without preculture, or to subtract recognized bacterial species from a microbiome to facilitate genomic analysis of the remaining species.
this approach has potential to be applied to different species in different and complex microbial communities.

 microbes are critical symbiotes for humans, where upwards of trillion foreign cells from more than different species reside. the gut is host to the bulk of the microflora, where bacteria are the most abundant, outnumbering eukaryotes and viruses by orders of magnitude. while a handful are known human pathogens, the majority of these bacteria, such as lactobacillus sp. are commensal or mutualistic, exerting their influence through probiotic functions. studies in mice and humans implicate gut bacterial influence not just in digestion of nutrients, but in fat storage, modulation of bone-mass density, angiogenesis, protection against pathogens, and immune functions. conditions such as crohn’s disease, diabetes, and obesity have all been directly linked to an imbalance of gut microflora. despite an explosion of research in recent years, the ecology and mechanistic details of complex microbiomes such as those found in the gut remain enigmatic, and new methodologies for dissection and characterization are needed. metagenomics refers to a powerful set of genomic and bioinformatic tools used to study the diversity, function, and physiology of complex microbial populations. substantial advances in microbiome research have been driven by the extensive use of next generation sequencing technologies, which allow annotation and characterization of microbiomes using targeted or shotgun approaches. targeted approaches are suboptimal in the identification of low abundant species, and even though identification of most species from a population is possible using shotgun sequencing, assembly of complete genomes of individual species is rarely possible unless those species are highly abundant. moreover, as complexity increases, dataset resolution decreases, reducing the ability to comprehensively analyze community structure. recent reports provide promising advances in metagenomic binning and assembly for the reconstruction of complete or near-complete genomes of rare community members from metagenomes. albertesen et al. have described differential-coverage binning as a method for providing sample-specific genome catalogs, while wrighton et al. have also been successful in sequencing more than 90% of the species in microbial communities. in another approach, either gc content or tetranucleotide frequency combined with genome coverage patterns across different sample preparations was used to bin sequences into separate populations, which were then assembled under the assumption that nucleotide frequencies are constant for any specific genome. sequencing throughput is continually improving and is expected to provide access to increasingly lower abundance populations and improvements in read length and quality will reduce the impact of co-assembly of closely related strains on the initial de novo assembly. while these approaches represent exciting advances in bioinformatic tools, experimental tools for reducing the complexity of a population prior to sequencing, such as enriching for low abundant organisms or intact cells, provide alternative and complementary approaches to improve genomic analysis of such complex systems. a variety of experimental methods have been used to decrease sample complexity prior to sequencing. the most commonly used tool for decreasing sample complexity is probably single cell genomics which utilizes flow cytometry, microfluidics, or micromanipulation to isolate single cells as templates for whole genome amplification by multiple displacement amplification. as it requires only a single template genome, it allows the sequencing of “uncultivable” organisms. for example, a recent paper from the quake group used microfluidics to isolate single bacterial cells from a complex microbial community, using morphology as discriminant, before genome amplification and analysis. scg approaches rely on mda, and while mda can generate micrograms of genomic amplicons for sequencing from a single cell, amplification bias, leading to incomplete genome coverage, is a major inherent limitation. in fact, a recent survey of genomes sequenced from single cells had a mean coverage of approximately 40%. a clever use of single amplified genome assembly improved coverage to >90% for of the genomes, with mean coverage being approximately 70% for the genomes when assembled from multiple sags. mda-associated amplification bias has been improved for eukaryotic cells using a technique called malbac, but these improvements have yet to be shown for prokaryotic genomes and still rely on random, or morphologically based, cell sorting. such random sorting of single microbial cells from complex mixtures is expected to bias against rare species and may require sorting and sequencing of hundreds to thousands of cells before a rare genome can be obtained. increased input template number can overcome mda amplification bias, or difficulties in processing and sorting single cells from biofilms, and provide near complete genome coverage. potential methods for accomplishing this include inducing artificial polyploidy or using gel microdroplets. however, in both of these cases, rare species may still be missed if sufficient numbers of single cells cannot be sorted. this has been partially addressed in a recently published “mini-metagenomics” approach. mda product coverage was improved by creating bacterial pools by flow cytometry, with ~ bacteria in each pool. screening of these pools for 16s rdna sequences of the bacterial species of interest, followed by deep sequencing of the positive pools, allowed assembly of a relatively complete genome from different pools containing the same 16s rna sequences. an alternative approach to simultaneously address both amplification bias and isolate rare species is to use antibodies recognizing specific microorganisms within microbial communities to enrich and/or subtract bacterial species prior to sequencing. we hypothesized that enrichment by selective sorting in this way could provide a powerful method for significantly increasing input template number to obtain complete genomes of low abundance species, akin to creating a small microbiome in which all members expressed a single target recognized by the antibody of interest. in the present work, we developed a selection and screening pipeline using phage display and flow cytometry to isolate a single chain fv antibody that can: i) identify a bacterial species, lactobacillus acidophilus, with extreme specificity; and ii) be applied to a microbiome, using fluorescence activated cell sorting, to identify, enrich, and deplete targeted species from bacterial mixtures. we further demonstrated that if this approach was applied to a mock community containing l. acidophilus, rather than the pure single species, antibodies recognizing l. acidophilus could be isolated. this phage display selection method is highly adaptable to recognition of any organism and provides a unique tool for dissection and sequencing of rare species from complex microbiomes.  selection against intact bacteria using phage display and screening by flow cytometry we chose the probiotic lactobacillus acidophilus atcc as a target for our approach. lactobacilli such as sp. acidophilus are widely studied gut microbes with probiotic functions including digestion, immune function, and prevention of diarrhea. antibody selections were performed against l. acidophilus using two methods. in the first, the bacteria were coated on immunotubes, while, in the second, selection was carried out by centrifugation. for each selection we used a previously described naïve scfv library displayed on m filamentous phage. two to three rounds of selection, with increasing stringency, were performed prior to re-cloning enriched scfvs into pep-gfp for screening. this vector generates scfv proteins in fusion with two different detection tags: sv recognized by a monoclonal antibody and s a split green fluorescent protein tag that fluoresces when complemented with gfp1-. the simultaneous use of both tags enhances signal-to-noise ratio when testing putative clones for binding activity against l. acidophilus in flow cytometry. scfv culture supernatant was incubated with l. acidophilus followed by staining and the l. acidophilus bacteria analyzed using an lsrii flow cytometer. sequencing revealed one unique scfv from the immunotube selection, and three unique scfvs from the selection by centrifugation. the α-la scfv was found to be highly specific for l. acidophilus, binding to all tested l. acidophilus strains, but not to a panel of other gut bacteria, including bifidobacterium sp., peptoniphilus sp., e. coli, and six different species of lactobacillus. our analysis included lactobacillus helveticus, the closest species to l. acidophilus, the 16s rrna sequence of which shares >98% identity. the other three α-la scfvs showed similar degrees of specificity. we proceeded with the α-la scfv for the remainder of the study due to greater expression and apparent affinity relative to the other α-la scfvs. the specificity of the α-la scfv was also further validated using the amnis image-stream mark ii flow cytometer, which captures microscope images in a flow cytometric configuration. lactobacillus strains were grown in atcc no. lactobacilli mrs broth. all other strains were grown in atcc no. reinforced clostridial broth with the exception of ec which was grown in luria broth. the specific surface antigen recognized by all the α-la scfvs was identified as the l. acidophilus s-layer a protein, using western blotting and mass spectrometry. slpa proteins are highly abundant, paracrystalline surface glycoproteins that make obvious targets for scfv recognition. further analysis following deglycosylation of the bacterium revealed that recognition was not mediated by glycosylation of the protein. scfv specificity to l. acidophilus in a mock community we tested the use of the isolated α-la scfv protein to detect varying abundances of l. acidophilus within a mixture of different bacterial species. we individually grew a total of ten species in their respective growth media. the various species were mixed to generate a “mock” community, which enabled us to control the relative composition of different species within the mixture. all species in the mock community were added at equal concentrations. the four resultant mock communities contained 10% of each of these species, and differed only in their relative abundance of l. acidophilus at 10%, 5%, 1%, and % in the community. staining with purified α-la scfv was followed by analysis by flow cytometry. pure l. acidophilus stained with α-la scfv was used to establish the l. acidophilus analysis gate as reference for varied l. acidophilus abundances in the mock communities. ten thousand events from each mock community were analyzed. we observed %, %, %, and % l. acidophilus in the mock 10%, 5%, 1%, and % communities, respectively. this degree of accuracy supports the possibility that the scfv can detect target bacteria within a population, with abundance less than %, and further supports the specific nature of the α-la scfv. targeted enrichment of single l. acidophilus cells from yogurt microbial community the ability to sort single l. acidophilus cells using the α-la scfv was subsequently tested on cultured yogurt, a natural, heterologous community the constituents of which are reported to include streptococcus thermophilus, lactobacillus delbrueckii subsp. bulgaricus, lactobacillus delbrueckii subsp. lactis, lactobacillus acidophilus, and bifidobacterium lactis. our aim was to validate specificity and test the ability of our selected scfv to recognize l. acidophilus from a culture even though the scfv was selected against bacteria grown in the laboratory. bacteria were isolated using methods previously described based on a series of density gradient centrifugations to remove sample debris prior to bacterial cell isolation. after staining with α-la scfv-gfp + α-sv5-pe, -5% of the total population, depending upon the yogurt preparation, fell into the l. acidophilus-specific gate. single bacterial cells were sorted from the pre-sort, negatively sorted, and positively sorted gates for amplification by mda and subsequent 16s rdna sequencing. we identified the species origin of individual cells sorted from four different replicates. the dominant species in the community was streptococcus thermophillus, with lactobacillus delbruekii and at least eight other species identified, including species that were not expected to be found in the yogurt culture. on average, sequencing showed l. acidophilus recovery at % in the pre-sort community, enrichment at % in p and complete absence in p, thereby demonstrating the feasibility of species depletion. in three of the replicates, l. acidophilus sequence was not observed in the pre-sort sample, but was nevertheless enriched and identified in the p gate, indicating that the l. acidophilus likely would not have been identified using standard single cell sorting and analysis. obtaining a complete genome using scfv targeted enrichment one of the primary goals of this study was to show that targeted enrichment of template using phage derived antibodies and facs can be used to generate complete genome sequences of rare species, with the specificity conferred by the selected scfv enabling the enrichment of enough template to complete a genome without any further downstream cultivation or chemical treatment prior to mda. to test this idea, l. acidophilus was sorted from one of the bacterial yogurt extractions, as either single cell or 50-cell templates for mda, and sequenced using the illumina miseq platform. for reference mapping, reads from both the single and 50-cell sorted amplicons were normalized and mapped to l. acidophilus ncfm. in parallel, as reference genomes are unavailable in most cases, we also assembled the genome de novo using the normalized reads. the assembly tool clc was used to both map reads and assemble contigs de novo. having a reference genome available allowed us to accurately assess the extent of genome coverage using both mapped reads and de novo assembly. as we hypothesized, reads mapping from the 50-cell template yielded near-complete genome coverage at %, while the single cell template fell short at 68% with far more amplification bias. bias is clear in the single cell template with a large portion of the genome lacking coverage while other regions are covered at very high frequencies of > fold. for the de novo assembled genome, the 50-cell template yielded contigs with > % coverage of the reference and ~8-10% contamination by sequences from non-l. acidophilus species. the contaminating non-lactobacillus reads were identified by searching assembled contigs in sequenced microbial genomes. we found that the single cell data was contaminated with sequences from bacteria close to a sequenced pseudomonas genome and the 50-cell data was contaminated with genomic sequences related to rhodopseudomonas, bradyrhizobium and nitrobacter. % of the single cell read data mapped to the pseudomonas genome and % of the 50-cell data mapped to the rhodopseudomonas genome, % to the bradyrhizobium and % to the nitrobacter. the contaminations were likely generated during the cell sorting and/or the mda process. mda-related contaminants, such as non-specific amplification and dna presented in reagents, are common to virtually any approach that utilizes whole genome amplification. beside possible contamination from the mda process, most contaminants were probably introduced during the cell sorting process since contaminated sequences were not shared between single and 50-cell results. we hypothesize that sorted specific cells may contain contaminating cells in the same droplet, or that contaminating dna, either free in solution or attached to the targeted cell may be sorted and become an mda template. we believe it more likely that the rhodopseudomonas genome, which was 34% covered, may have been introduced by cell contamination, while lower level contamination may have occurred via the second mechanism. fortunately, the vast majority of contaminant reads was easily removed and did not interfere with full data analysis of assembled contigs. to assess coverage, de novo assembled contigs were mapped back to the reference and the resulting coverage was > % for the 50-cell template and 63% for the single cell. these values are highly similar to those expected from draft coverage of cultured bacteria, indicating that template number enrichment using specific scfvs and facs can be used to sequence very low abundance genomes in a community once a specific antibody is available. selecting antibodies against a mock community to determine whether this method can be applied to more complex microbial communities, we selected phage antibodies against the mock community used above, with each bacterial species present at ~10%. selection was carried out by centrifugation, and after two rounds, the heavy chain complementarity determining region of the complete antibody output was sequenced by ion torrent. the hcdr is the most diverse cdr, contributes most to antibody binding specificity, and is widely used as a surrogate for vh and scfv identity. using the antibody mining toolbox, the hcdr3s of the antibodies selected against the mock community were identified and ranked for abundance. as shown in table  three of the twenty most abundant antibodies had hcdr3s that were identical to three of the previously selected antibodies recognizing l. acidophlius, indicating that, in principle, it may be possible to select species specific antibodies directly against individual bacteria in complex bacterial communities, without the need to culture the individual bacteria. however, validation of this possibility will require additional experimentation and selection on natural microbiomes rather than the mock community used here. l. acidophilus *total number of quality reads from the ion torrent sequencing run = , discussion the expanding field of metagenomics continues to search for robust ways to obtain high-quality genomes from under-represented or rare species in a given sample. improvements in sequencing throughput will enable access to lower abundance populations, but a “pre-enrichment/pre-clearing” step before the analysis can provide complementary and significant results. we describe a novel and adaptable approach for sequencing low abundance genomes from microbial communities, with potential improvements in the genomic coverage of low abundance species where standard single cell approaches result in incomplete genomes or may have missed the organism altogether. we demonstrate the use of phage display to select antibodies against a bacterial species with exquisite specificity. the use of in vitro display potentially allows the method to be adapted to any organism or microbiome, does not rely on commercially available antibodies, and generates antibodies that are highly renewable and amenable to further engineering to modify affinity or specificity. to demonstrate the feasibility of the approach, we first targeted lactobacillus acidophilus, a bacteria naturally found in environmental samples from food to feces and is a principal commensal bacterium of the human gut. the tested α-la scfv proved to be extremely specific and did not recognize other common gut microflora. while it is practically impossible to prove that this scfv does not recognize any other bacteria, when tested on other lactobacilli such as l. helveticus, which is highly similar to l. acidophilus, we did not observe binding, providing strong evidence that the scfv is species-specific. the target protein recognized by our scfv was identified as the surface layer protein a. s-layer proteins are highly abundant and ubiquitous crystalline surface structures that have been implicated as a principal component for the organism’s probiotic functions. other lactobacilli tested in this study produce s-layer proteins that are highly similar, but which can nevertheless be distinguished by our α-la scfv, demonstrating the high degree of specificity achievable. since s-layer proteins are common to many bacteria, future work may involve re-engineering the α-la scfv to target s-layer proteins from other organisms, an option that is only possible with in vitro derived antibodies. coupling the specificity of phage-selected α-la scfv with facs allowed precise manipulation of a population on a per-cell basis, making possible the sufficient enrichment of l. acidophilus for > % genome coverage using both reference mapping and de novo assembly. while it is common to observe this level of coverage for de novo assembly when the target organism is cultured prior to sequencing in the laboratory, the level of coverage reported here for a bacteria extracted from an environmental sample is exceptional. for sequencing, we easily and rapidly sorted 50 l. acidophilus cells from an environmental sample where l. acidophilus comprised ~ % of the population and were able to rapidly detect and quantify l. acidophilus at ~ % in a mock community comprising nine other species. although we only tested compositions as low as ~ %, we are confident that l. acidophilus could be identified from mixtures where it is even lower in relative abundance with detection limited solely by the total number of cells available in a mixture and time available for sorting. while detection and enrichment of rare species is an obvious use of these antibodies, depletion of common species may be equally important, as bias towards high abundance species is a well-known issue when performing shotgun metagenomics and, potentially, non-targeted single cell genomics. our single cell analysis shows that l. acidophilus is completely depleted from the sample in the negative sort gate, demonstrating the feasibility of both depletion and enrichment. separation methods, namely immunoprecipitation, micromanipulation, and flow cytometry have been described to improve genome sequencing, and the approach described here may also be applicable to other microbes found in microbiomes without being limited to organisms with innate fluorescence, distinct morphology and/or high genome copy number. in this study we generated a scfv against an organism that can be cultured in the lab as a demonstration that recombinant antibodies can be raised against a specific organism and used to dissect, phylotype, and recover complete genomes for organisms from microbial communities. we used an organism with a reference genome in order to accurately assess genome coverage. future studies will involve selecting antibodies directly against uncultivable organisms within complex microbiomes. we provide proof of principle, using selection against a mock community, that such an approach is potentially feasible: hcdr sequences of three of the antibodies selected against the pure culture were identical to those of antibodies selected against the mock community. while this is promising, it is likely that selection procedures will have to be modified in order to select antibodies against the many different species present in a natural microbial community. in particular, we have previously shown that selection against a specific antigen is far more efficient when carried out against the individual antigen than when the antigen is present in a mixture of other antigens. the situation is likely to be even more challenging for microbial communities, and may require selection in emulsions, microfluidics or against individual cells to ensure that individual bacteria are isolated from one another during the selection process. if the identity of the recognized bacteria in the microbiome is unimportant – i.e. the goal is to catalog genome sequences present in a microbiome, whatever they are – the use of this method may be relatively straightforward. it is likely to be more challenging, however, if the goal is to select antibodies against particular species in a population, unless an alternative means of bacterial isolation, such as fluorescent in situ hybridization, is available. one possible approach, which may be successful in microbiomes comprising few species, would be to select a panel of positive antibodies against different species within the community, and then deconvolute species recognition using facs and deep sequencing in a manner similar to that described here, after antibody selection and sorting. however, the number of bacteria that can be extracted from environmental samples easily exceeds the number required for phage selection suggesting that this approach will be difficult for more complex populations. since depletion is as feasible as enrichment using these scfvs with facs, it may be possible to iterate the process using scfvs against high abundance species for their subtraction and, thus, enrich for the low abundance organisms. even if antibodies cannot be raised to low abundance organisms, depletion of high abundance organisms in a mixture will concentrate the low abundance ones, and so lead to improved taxonomic identification and genome recovery. the described approach also has potential not only for the genome sequencing of novel and uncultivable organisms, but also in comparative genomics. in this regard, selection of antibodies against organisms initially grown in the lab then used on environmental and clinical samples holds great potential for medicine and epidemiology. for example, a recent study reports the use of a commercially available igg antibody for targeted enrichment using immunomagnetic separation to fully sequence chlamydia trachomatis directly from clinical isolates without culture. our approach could extend on this work by adding a mechanism for the initial selection of suitable antibodies for studying pathogenic, probiotic, or other organisms. near complete coverage, such as that provided by enrichment with phage-selected scfvs, is paramount for high resolution genomic comparisons. in fact, while a discussion of genome differences is outside the scope of this study, we observed at least single nucleotide polymophisms when comparing the extracted l. acidophilus to the reference genome showing that the α-la scfv reported here could be used immediately for future comparative genome studies on human-derived l. acidophilus for both research and clinical purposes.  in this paper we demonstrate the power of combining phage antibody selection directly on bacteria with fluorescence activated cell sorting and deep sequencing to either enrich, or deplete, bacteria recognized by specific selected antibodies. using this approach it becomes possible to assemble genomes directly from complex microbiomes without preculture, or to subtract recognized bacterial species from a microbiome to facilitate genomic analysis of the remaining species. this approach has potential to be applied to different species in different and complex microbial communities. 
we selected antibodies against lactobacillus acidophilus and demonstrate a facs-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. phage antibodiesgenome completionsingle cell genomicsmdaflow cytometry we describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting. single chain antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in facs for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. the ability to selectively enrich for l. acidophilus when it is present at a very low abundance leads to complete de novo genome coverage whereas the standard single-cell sequencing approach is incomplete. we show that specific antibodies can be selected against l. acidophilus when the monoculture is used as antigen as well as when a community of closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. the approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. combining antibody specificity with facs provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.
 single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. methods we describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting. single chain antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in facs for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing.  we selected antibodies against lactobacillus acidophilus and demonstrate a facs-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. the ability to selectively enrich for l. acidophilus when it is present at a very low abundance leads to complete de novo genome coverage whereas the standard single-cell sequencing approach is incomplete. we show that specific antibodies can be selected against l. acidophilus when the monoculture is used as antigen as well as when a community of closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities.  the approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. combining antibody specificity with facs provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing. phage antibodiesgenome completionsingle cell genomicsmdaflow cytometry 