The sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging.To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated.We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.The online version contains supplementary material available at 10.1186/s12864-021-07781-1. This regulation of transcript sequence content through alternative polyadenylation (APA) occurs in the majority of genes in yeast, plant, and mammalian genomes [During the co-transcriptional processing of a pre-mRNA, the 3′ end of the transcript is cleaved and a polyadenine tail is added that promotes the stability and translation of the resulting message , 2. The  genomes .The cleavage and polyadenylation reaction is performed by the core CSTF and CPSF complexes and CFIm which associate with RNA polymerase II (Pol II) transcription complexes , 8 and tRegulation by these factors results in the large variation in 3′ UTR content seen across tissues and developmental stages . Specifi, top). The second, which we will refer to as “alternative last exons” or “ALEs”, occurs when multiple APA sites are found within different terminal exons . Regulation of the choice between alternative tandem UTRs can be viewed as a competition between a proximal upstream poly(A) site that is transcribed first with a distal downstream site that is transcribed second. Similarly the choice between ALE’s can be viewed as a competition between recognition of a proximal 5′ splice site and a distal poly(A) site [Alternative polyadenylation exists in two structurally distinct forms. The first, which we will refer to as “tandem UTRs”, occurs when multiple APA sites are found within the same terminal exon Fig. B, top. Tons Fig. B, bottom(A) site . It is n(A) site , 23.FigThe majority of published high-throughput RNA sequencing data has been produced using libraries in which the entire transcript is represented. Although these libraries are informative for the regulation of processes like alternative splicing, they are not ideally suited for the quantification of APA isoforms. Alternative library preparation strategies that specifically profile the 3′ ends of transcripts, including 3′ end sequencing and 3′ READS , 25, likThe study of APA using high-throughput RNA sequencing has been facilitated through a handful of software packages aimed at quantifying changes in relative APA site usage across conditions –29. HoweTo take advantage of this advance in isoform quantification and apply it to the analysis of APA isoforms, we developed LABRAT (Lightweight Alignment-Based Reckoning of Alternative Three-prime ends). A particular advantage of our approach is that it permits rapid analysis of large numbers of publically available RNA-seq data sets including patient samples. Here, we applied this approach to tens of thousands of RNAseq samples to study processes and factors that regulate relative APA isoform abundance as well as the consequences of APA site choice on transcript fate.To quantify relative alternative polyadenylation site usage from RNAseq data, LABRAT takes a genome annotation file and first searches the annotation for tags that define transcripts with ill-defined 3′ ends in order to filter and remove them from further analysis Fig. A. Becausm, which is defined as its position within this proximal-to-distal ordering, beginning with 0. Each gene is assigned a value, n, which is defined as the number of distinct APA sites that it contains. The expression (TPM) of every terminal fragment belonging to a given APA site is then summed to define the expression level of the APA site, and this process is repeated for every APA site within a gene. The expression level of each APA site is then scaled according to the following formula:For each gene, alternative polyadenylation sites are then defined using terminal fragments. Terminal fragments with 3′ ends within 25 nt of other 3′ ends are grouped together to define a single polyadenylation site, and the sites are ordered from most gene-proximal to most gene-distal. Each APA site within a gene is assigned a value, ψ. Scaled and unscaled TPM values are summed across all APA sites within a gene, and ψ is defined as the ratio between these summed values:To quantify a gene’s relative APA site usage, we defined a term, ψ value of 0, while those that show exclusive usage of the most gene-distal APA site will be assigned a ψ value of 1 . To further explore changes in ψ value for specific genes, we plotted read coverages over two genes that showed significantly more downstream APA site usage in brain tissue: Slc16a7 and Elavl1 . For both genes, we observed significantly lower read coverages corresponding to usage of the distal APA site in the liver samples relative to the brain samples. Accordingly, LABRAT assigned these genes to have low ψ values in the liver samples, and high ψ values in the brain samples, indicating that LABRAT can accurately quantify APA.We found 470 genes that displayed differential relative APA isoform abundance between the tissues (FDR < 0.05) Fig. C. As expFigureB). In this analysis, brain and testis samples were clear outliers. Interestingly, performing the PCA analysis using only tandem UTR  or ALE  genes produced very similar results, suggesting that these two forms of APA are broadly coregulated across many tissues.To perform similar analyses in human samples, we analyzed over 5000 RNAseq samples from over 30 different human tissues produced as part of the Genotype-Tissue Expression (GTEx) project . We quanp < 2.2e-16) . Conversely, testis samples exhibited a similar bias for the use of upstream APA sites (p < 2.2e-16) . The propensity of testis to use upstream APA sites has been previously observed [To understand more about APA in human brain and testis, we compared their APA profiles to those observed in human liver samples. As expected, we observed that brain samples exhibited a significant bias for the use of downstream APA sites  and compared them to values produced by the more classical approach of simply counting the aligned reads associated with each APA site (see Methods). We found that the ψ values produced by LABRAT were in strong agreement with those produced by alignment-dependent method (R ~ 0.92) . It is important to note here that the deviation from perfect agreement is possibly due to the greater ability of Salmon (and therefore LABRAT) to accurately assign reads that could be consistent with multiple polyadenylation sites.To assess the accuracy of APA isoform quantification with LABRAT from 3′ end data, we used a dataset in which the authors prepared 3′ end libraries from RNA isolated from human peripheral blood mononuclear cells (PBMCs) with and without treatment with poly dI:dC . Salmon,RAT Fig. E,S1G anψ values from the RNAseq and 3′ end data using ‘RNAseq’ and ‘3pseq’ librarytype parameters, respectively. We found that ψ values from the two library preparation methods were reasonably and reproducibly correlated (R ~ 0.67) while ψ values from samples produced using the same highly correlated (R ~ 0.97)  Fig. F. RNAseques Fig. F, givingTo compare LABRAT with other APA analysis tools, we generated a synthetic RNAseq dataset containing 50 million reads in which 1250 genes displayed increased distal APA site usage, 1250 genes displayed increased proximal APA site usage, and 2500 genes displayed no change in APA site usage . We usedψ values calculated by LABRAT were highly correlated to the analogous metric used by QAPA, PPAU (R = 0.81) . In comparing the four methods, LABRAT was the best suited to accurately identify differential APA in the simulated data .QAPA, like LABRAT, uses lightweight alignments to quantify APA. Reassuringly, we found that U R = 0.8 , finding between 10 and 740 genes that fit this criterion in each sample . For these genes, we then compared their ψ values across compartments by subtracting the ψ value in the cell body from the ψ in the projection to define Δψ. Genes with positive Δψ values therefore had their distal APA isoform enriched in projections while those with negative Δψ values had their proximal APA isoform enriched in projections.For all samples, we identified genes whose ple Fig. A. Many oΔψ values, indicating a broad connection between the use of distal APA sites and localization of the resulting transcript to cell projections  between the fraction of ALE genes with positive Δψ values and the fraction of tandem UTR genes with positive Δψ values  cells, human HepG2 cells, and human K562 cells [To further explore connections between APA and RNA localization beyond cell projections, we used LABRAT to analyze RNAseq data from a biochemical fractionation of 3 cell types, 62 cells . In thesψ values for genes across cellular compartments. Hierarchical clustering of samples based on ψ values revealed that samples from the same fraction generally clustered with each other, indicating the high quality of the data . To minimize the effect of library preparation on the identification of genes with significantly different ψ values across compartments, we included the library preparation method as a covariate in LABRAT’s linear model. This allowed us to pool all of the samples for a given compartment in order to identify genes with significantly different ψ values between compartments regardless of library preparation method.As with the projection data, we compared ψ values across any pairwise comparison between cytosolic, membrane-associated, and insoluble fractions (FDR < 0.05). Based on our observations relating distal APA and RNA localization to projections, we then asked if any of these fractions were associated with higher ψ values than the other two. We visualized these comparisons using simplex plots , indicating a preferential association of downstream APA isoforms with that fraction.We first identified genes with significantly different ots Fig. C. In theion Fig. C,S2E, Fψ values between the fractions (FDR < 0.01). Of these, 492 (89%) had a ψ value that was higher in the membrane fraction than the cytosolic fraction, indicating a broad association between transcripts produced using distal APA sites and the membrane fraction .Because of the apparent bias toward distal APA site use among membrane-associated transcripts, we next focused on comparing the cytosolic and membrane fractions. When comparing the cytosolic and membrane fractions of HepG2 cells, we identified 552 genes that had significantly different Δψvalues (membrane - cytosol) for all genes expressed in both cell lines. We observed a strong correlation (R = 0.73) between Δψ values in the two cell lines  regulates multiple co-transcriptional processes, including alternative splicing, and termination that is coupled to poly(A) site cleavage , 53–56. During transcription, a gene-proximal APA site is necessarily transcribed before a gene-distal APA site. There exists a time, therefore, during which the proximal site is the only APA site that exists on the transcript. Reducing the speed of Pol II transcription would increase this time in which the proximal site is free from competition with the distal site. We hypothesized that this would lead to an increase in usage of the proximal APA site Fig. A. IndeedIf the shift in APA was related to the amount of time during which the proximal site was exclusive, then the shift should be most pronounced in genes in which the distance between proximal and distal sites is large. Consistent with this hypothesis, we found that this “inter-polyA distance” for genes that displayed increased proximal APA was significantly longer than expected Fig. D, furtheψ value from the wildtype Pol II sample alone (R = 0.01). However, in this analysis across genes, other factors that influence APA (e.g. the relative strengths of upstream and downstream polyadenylation sites) can dominate. Comparing within genes but across conditions, as done above using Δψ, alleviates this concern.We found no correlation between a gene’s inter-polyA distance and its If alternative polyadenylation of tandem UTRs and ALEs were generally coregulated, then it would be expected that changes in Pol II speed would affect both classes of genes. To test this, we examined the increase in proximal APA site usage caused by slow transcription in the context of tandem UTR and ALE genes separately. We found that proximal APA usage was increased for both tandem UTR and ALE genes Fig. E, F, indψ values for all expressed genes between RBP knockdown and control knockdown samples for 191 RBPs that were expressed in both cell lines. To identify genes that had significantly different ψ values (FDR < 0.05) between RBP knockdown and control knockdown samples, we incorporated the cell line of the experiment as a covariate in LABRAT’s linear model.To investigate the contributions that individual RBPs can have to the regulation of APA isoform abundance, we analyzed the ENCODE RBP knockdown RNAseq datasets with LABRAT , 58. ThiΔψ values (control knockdown - RBP knockdown) for all expressed genes in a given RBP knockdown in HepG2 cells with their Δψ values upon knockdown of the same RBP in K562 cells. We therefore end up with one correlation coefficient per RBP knockdown. As a control, we compared these values to correlations of Δψ values where the RBP that was knocked down was different between the cell lines . When we restricted the comparison to genes that had significantly different ψ values between RBP and control knockdowns (FDR < 0.05), we observed a much higher correlation of Δψ values between cell lines  , and of these 52 had α values of greater than 0.5 while 42 had α values less than 0.5 . Fip1 has been found to promote proximal APA isoforms [α = 0.39, p = 0.007). Similarly, CSTF2 has been noted to promote proximal APA isoforms [α = 0.31, p = 0.0001). These LABRAT analyses reflecting prior literature on specific RBPs gave us increased confidence in the ability of LABRAT to probe RBP-specific effects on APA isoform abundance.The effects of specific RBPs on APA isoform abundance have been reported for a handful of RBPs. CPSF6 has been found to promote distal APA isoforms , 13, andisoforms . While Fisoforms . While Cα values for tandem UTR and ALE genes separately. α values for these two APA types were highly correlated (R = 0.62), further indicating that these two mechanisms of APA regulation are not independent and share elements in common .For each RBP knockdown experiment we then calculated FiguresB, C).If changes in APA isoform abundance upon RNAi were directly due to loss of the RBP, then we would expect that the RBP would directly bind the 3′ UTRs of the genes whose APA isoform abundance it regulates. To test this, we analyzed RBP/RNA interactions as measured by the eCLIP experiments performed as part of the ENCODE project . We obseψ value upon RBP knockdown also contained an eCLIP peak for that RBP in their 3′ UTR. We then calculated whether this overlap of RBP binding and function was statistically significant . For 21 of these RBPs, we observed a significant overlap between the RBPs functional APA isoform regulatory targets and the 3′ UTRs it bound .In HepG2 cells, 84 RBPs had both RNAseq data from RNAi experiments and eCLIP data. For each RBP, we calculated how many of the genes with significant changes in p = 0.4) , overall, these results indicate that many of the RBPs tested are modulating relative APA isoform abundance through direct interactions. We then repeated the analysis, but looked for eCLIP peak overlaps throughout the gene bodies of APA targets . In this analysis, we did not observe that RBPs were preferentially bound to their APA isoform regulatory targets. Taken together with the results of the 3′ UTR-centric analysis, these results indicate that RBPs that regulate relative APA isoform abundance likely do so through binding the 3′ UTRs of their targets.Although we did not observe a similar significant relationship between APA and eCLIP data in K562 cells  between tumor and normal samples. We then defined Δψ values  to ask whether proximal or distal sites showed increased usage in tumor samples. For some cancers, including Lung Squamous Cell Carcinoma (LUSC), Urothelial Bladder Carcinoma (BLCA) and Lung Adenocarcinoma (LUAD), tumors displayed the expected pattern of increased proximal APA in tumors  in which the ψ value was greater in the tumor sample than the normal sample. Put another way, for each patient, we calculated the fraction of significant tandem UTR and ALE genes with positive Δψ  values  for each cancer were compared. We found that for head and neck squamous cell carcinoma (HNSC), a cancer that typically exhibits increased proximal APA, patients with lower ψ values in misregulated genes had poorer prognoses (p = 0.0069) compared to patients with higher ψ values for the same genes   Fig. E. Therefψ and changes in RNA levels across the 191 ENCODE RBP knockdown sample pairs and the 671 TCGA tumor/normal sample pairs. To do so, we defined a term, rho (ρ), as the correlation between changes in ψ and changes in gene expression across two samples . Relaxing the transcript expression threshold used by LABRAT from 5 TPM to 1 TPM had a minimal effect on these results as the median ρ value remained negative .It could be the case, though, that for specific genes, APA and gene expression may be more strongly linked. To explore this, we calculated irs Fig. D, E. Theρ value distributions were long, indicating that there were genes whose changes in ψ value and changes in expression were highly correlated across conditions. We selected three of these, RPLP1, NOLC1, and UBE2G1, for further analysis. Given that each of these genes had strong negative ρ values in both the ENCODE and TCGA data  gene sets . These gene sets behave differently: positively correlated genes are more highly expressed when downstream PAS are used (increased ψ) while negatively correlated genes become less expressed as they utilize more downstream PAS.To better understand sequence elements downstream of proximal APA sites that may reduce RNA expression, we used the This analysis was simplified by only considering genes with two APA isoforms such that RNA expression could be explained by proximal or distal UTR usage. The analyzed UTR sequences were unique, meaning that tandem UTRs were separated into proximal and distal UTRs such that distal UTRs lacked their shared 5′ sequence Fig. H. This a. This increased 3′ UTR length may make these isoforms more susceptible to NMD, partially explaining their decreased expression [. Conversely the distal UTRs of positively correlated genes were depleted for AU-rich five-mers . Unsurprisingly given their AU-richness, negatively correlated genes were enriched for ARE binding protein motifs in their distal UTRs and contained more AREs as scored by AREScore [. AREs are destabilizing RNA elements bound by several ARE binding proteins that facilitate RNA degradation. The presence of AREs in distal UTRs of negatively correlated genes is consistent with lower RNA expression when downstream PAS are utilized. It is important to note that the distal UTRs of positively correlated genes are depleted for AREs consistent with their higher expression. These results suggest that APA can regulate gene expression through the inclusion of destabilizing AREs in a transcript’s 3′ UTR. Further, given how these results mirror previously observed effects of 3′ UTR AREs [Negatively correlated genes were found to have longer distal UTRs with lower GC content than expected Fig. J, K. Thipression , 68. AddAREScore    Fig. A. RBPs wα and β values for RBPs. We restricted this comparison to the 94 RBPs that had α values significantly different from the expected value of 0.5 . For these RBPs, α and β values were positively correlated . RBPs with α values greater than 0.5 had significantly higher β values than those with α values less than 0.5  between these two measurements, indicating that changes in their expression across diverse samples are controlled in large part by modulation of APA site choice.Relationships between APA and gene expression have also been well documented , 20. EarAcross over a thousand pairs of samples, we observed strong correlations between APA changes in genes with tandem UTRs and those with ALEs. If a particular condition promoted increased distal APA in tandem UTR genes, it overwhelmingly also promoted increased distal APA in ALE genes and vice versa. This strongly indicates that the two may be regulated by similar mechanisms, and hints of this connection have been observed before . Tandem Overall, the results presented here shed light on the molecular consequences of APA and make predictions about the proteins and mechanisms involved in its regulation. Further experimental studies are needed to fully understand these processes. We envision LABRAT as an important tool in deriving meaningful insights from those experiments.https://github.com/TaliaferroLab/LABRAT/. LABRAT searches for specific tags (mRNA_end_NF) in the annotation associated with transcripts with ill-defined 3′ ends. Optionally, LABRAT may also filter out transcripts that are not protein-coding (by looking for the ‘protein_coding’ tag. This may help remove transcripts that are not fully processed and therefore still nuclear. These tags are present in Gencode (www.gencodegenes.org) gff annotations but may not be present in annotations from other sources. For this reason, we strongly suggest using Gencode annotations for use with LABRAT. For analysis of Drosophila data, we modified LABRAT to perform similar filtering on Ensembl annotations for the dm6 Drosophila genome build. However, in principle, LABRAT can work with any annotation, including those that make use of 3′ end sequencing data to identify polyadenylation sites, so long as the polyadenylation sites are incorporated into a transcript model. This version of LABRAT is also available at the above GitHub address.LABRAT is freely available for download here: ψ value of NA.Genes that did not pass an expression filter (TPM ≥ 5) were removed from further analysis. This gene expression was defined as the sum of the expression values for all valid, filter-passing transcripts for the gene. LABRAT reports these genes as having a ψ values across conditions was done using a linear mixed effects model with the Python package statsmodels [ψ values was used . For analysis of the CeFra and ENCODE data, slightly more complex models were used. In the CeFra data, the method of library preparation, polyA-enrichment or ribosomal RNA depletion, was added as a covariate . In the ENCODE data, the cell line, K562 or HepG2, was added as a covariate . These models were then compared to null models where the effect of the condition was removed. For simple comparisons, the null models were specified as . For the CeFra and ENCODE comparisons, these were specified as  and , respectively. A likelihood ratio test was then used to evaluate the relative fit between the experimental and null models. P values were derived from the likelihood ratio test and then corrected for multiple hypothesis testing using a Benjamini-Hochberg correction [Δψ values are defined as differences in mean ψ across conditions.Identification of genes with significantly different tsmodels . For simrrection . Δψ valuTo define tandem UTR and ALE structures, LABRAT observes the isoform structures at the 3′ end of a gene. If all APA sites are contained within the same exon, then the structure in tandem UTR. If all APA sites are contained within different exons, then the structure is ALE. If a gene has only two APA sites, then its structure must be either tandem UTR or ALE. If a gene has more than two APA sites, it is possible for the gene to fit into neither classification. For example, in a gene with three APA sites, it is possible to have two of them contained within one exon and the third by itself in another exon. In these cases, LABRAT assigns the gene to have a “mixed” structure.https://github.com/daler/gffutils/). This process can take a few hours, depending on the size of the annotation. However, it only needs to be completed once. All future runs will automatically make use of a database file written after the indexing completes. Importantly, if indexing is interrupted, this file will still be written, and LABRAT will attempt to use this truncated file in the next run. This will cause problems. To prevent this, if indexing is interrupted, be sure to delete the resulting database file. It can be found at the location of the gff annotation, and ends with ‘.db’.If LABRAT is encountering a gff genome annotation file for the first time, it indexes this file using gffutils  [ψ values were identified as those with an FDR of less than 0.05.RNAseq data for mouse brain and liver tissues was downloaded from (A375882) . Each tiψ values were calculated for each gene in each sample using LABRAT. LABRAT employs an expression level cutoff, returning a ψ value of NA if the sum of expression of all isoforms for a gene is not at least 5 TPM. There were many genes in this analysis of tissue-specific RNAseq that therefore had ψ values of NA in at least one sample. To facilitate PCA analysis, these missing ψ values were imputed using the R package missMDA [RNAseq data from the Genotype-Tissue Expression (GTEx) project (BioProject PRJNA75899) were downloaded from the NCBI Sequence Read Archive (SRA) via dbGaP-authenticated access and quantified using salmon  as descr missMDA .phs000424.vN.pN between 07/16/2020 and 08/31/2020.The data used for the analyses described in this manuscript were obtained from dbGaP accession number ψ values from 3′ end sequencing data, we first trimmed 12 nucleotides from the 3′ of the reads as suggested by the authors that produced the data [ψ values from this data using LABRAT, we added the ‘librarytype’ parameter. If this value is set to ‘3pseq’, LABRAT will use Salmon-quantified counts for APA abundance estimation instead of length-normalized TPM values.To quantify the data . To calcThese 3′ end sequencing libraries were produced using the Quantseq FWD strategy (Lexogen). The single end reads produced by this approach correspond to the end of the fragment opposite of the oligo dT anchor. The beginning of this read is therefore one fragment length away (approximately 200–300 nt) from the polyA tail. Because of this library design, the majority of reads associated with a polyA site in 3′ end sequencing data should lie within 300 nt of the polyA site. Therefore, in contrast to the LABRAT RNAseq approach where the final two exons of every transcript are used for quantification, the LABRAT 3pseq approach quantifies the last 300 nt of every transcript.ψ values from 3′ end sequencing data using counts of aligned reads, we first trimmed 12 nucleotides from the 3′ of the reads as suggested by the authors that produced the data [To calculate the data  and thenthe data .https://github.com/TaliaferroLab/LABRAT/blob/master/countfrombam.py) to count the number of aligned reads associated with each polyA site. This was defined as the number of reads in the 300 nt preceding the polyA site. From these counts, ψ values were calculated by combining reads across all of the transcripts with a common polyA site, scaling the counts according to the position of the polyA site within the gene, and computing the ratio of scaled counts to unscaled counts.We then wrote a custom Python script  made by the software were compared to the assignments made during preparation of the synthetic dataset. For analysis of these reads with LABRAT, genes with FDR values of less than 0.05 were called as affected genes  while those with values of 0.05 or greater were called as control genes. For analysis with QAPA, genes with differences in PPAU values of at least 10 were called as affected genes while those with differences in PPAU values of less than 10 were called as control genes. For analysis with DaPars, genes with adjusted p values of less than 0.05 were called as affected genes while those with adjusted p values of 0.05 or greater were called as control genes. For analysis with Roar, genes with p values less than 0.05 and roar values greater than 1.1 were called as positive Δψ genes, genes with p values less than 0.05 and roar values less than 0.9 were called as negative Δψ genes, while genes with p values of 0.05 or greater were called as control genes.In analyzing the reads with each package, gene assignments  as a covariate.For each gene, the translation of its longest CDS sequence was given to the signal sequence prediction program SignalP . For a sψ values between wildtype and R749H samples were identified using LABRAT.RNAseq data from HEK293 cells expressing slow (R749H) and wildtype RNA polymerase II  were dowψ values were calculated comparing RBP knockdown and control knockdown samples, and genes with significant ψ differences between RBP RNAi and control RNAi samples were identified using an FDR cutoff of 0.05. FDRs were calculated using a linear model that incorporated the cell line (HepG2 or K562) as a covariate.In this dataset, each RBP was associated with two RBP RNAi samples and two control RNAi samples. We limited analyses to RBPs that had knockdown samples in both K562 and HepG2 cell lines. ψ values in the control RNAi than RBP RNAi was calculated. These fractions were defined as a value, α, where α ranged from 0 to 1. α values greater than 0.5 were therefore associated with larger ψ values  in the control RNAi sample. Conversely, α values less than 0.5 were therefore associated with smaller ψ values  in the control RNAi sample. Each RBP was therefore assigned one α value from the ENCODE data. Binomial p values were calculated for deviations from the expected fraction of 50%.For each RBP, the fraction of these significant genes with greater www.encodeproject.org. Analyses were restricted for within each line and not combined. For each individual RBP data set, overlapping peaks were merged using bedtools v2.29.2 [The eCLIP narrowpeak bed files for isogenic replicates aligned to GRCh38 for each RBP measured in both HepG2 (103 RBPs) and K562 (120 RBPs) were downloaded from  v2.29.2 . These pThe number of RBPs that were ‘self significant’, i.e. the occupancy of a specific RBP was significant for the genes whose polyA site choice was affected by knockdown of that same RBP, was determined for both HepG2 and K562. To determine if that number was greater than what was expected by chance, relationships between RBPs and the genes they bind were shuffled, and the analysis was repeated to identify the number of ‘self significant’ RBPs. This process was repeated 1000 times to generate a null distribution of the number of ‘self significant’ RBPs. The number of actual ‘self significant’ RBPs was then compared to the null distribution and an empirical p value was calculated.ψ values were calculated for each sample, and genes with significant ψ differences between all tumor samples and all normal samples within a cancer type were identified using an FDR cutoff of 0.05.In this dataset, each patient is associated with a pair of samples, one from a tumor and another from matched normal tissue. ψ was inferred by correlating changes in the RBP’s expression across samples with changes in ψ values of genes that passed the FDR cutoff of 0.05. For each tumor/normal pair, the change in RBP expression was calculated by comparing TPM expression values, and changes in ψ were calculated by finding the median Δψ value across genes with significant changes in ψ. The spearman correlation coefficient of this comparison across all tumor/normal pairs was defined as β. Each RBP was therefore assigned one β value from the TCGA data.Using the TCGA data, the effect of an RBP’s expression on ψ differences (FDR < 0.05) were identified for each tumor type as misregulated genes. The median ψ of misregulated genes was then calculated for each patient in samples without matched normal tissue controls. Patients were then ranked by their median ψ of misregulated genes and separated into quartiles. Only patients within the most extreme quartiles were plotted for each tumor type.Using the tumor and matched normal tissue samples from the TCGA dataset, genes with significant Clinical data for each patient was obtained from cbioportal . Survivaψ value for every gene was calculated. Gene expression filters (TPM ≥ 5) were applied, but FDR cutoffs for Δψ were not. These two values were then compared to each other, and the resulting Spearman correlation coefficient was defined as rho (ρ). If distal APA (i.e. increases in ψ) was associated with decreases in RNA expression, the resulting ρ value would be negative.For every pair of samples , the change in RNA expression and ρ was calculated in two different ways. In the first way, changes in expression and ψ for all genes within a sample were correlated. In this comparison, each sample pair ends up with a single ρ value. In the second way, changes in expression and ψ for a single gene across all sample pairs were correlated. In this comparison, each gene ends up with a single ρ value in each sample set (ENCODE and TCGA).ρ calculations were used to categorize genes as being either positively or negatively correlated. To achieve similar numbers of genes in each category, a positive ρ in either sample set was considered as positively correlated while a ρ less than − 0.15 in either sample set was considered negatively correlated. Genes behaving inconsistently between sample sets were removed from these categories and placed in the control gene category (25% of positively correlated and 14% or negatively correlated). For simplicity, genes with only two APA isoforms were considered during this categorization resulting in 316 positively correlated genes, 313 negatively correlated genes and 1466 control genes used in UTR sequence analysis.The second Proximal and distal UTR regions were cloned onto the coding sequence of Firefly luciferase. In this plasmid, Firefly luciferase is driven by a bidirectional tet-On promoter. This promoter also drives Renilla luciferase, which served as a control in these experiments. The resulting plasmids were transfected into HeLa cells using Lipofectamine 2000 (Life Technologies). These cells were engineered to contain a single loxP-flanked cassette within their genome . The plaThe expression of Firefly and Renilla luciferase transcripts was induced by incubating cells with 1 μg / mL doxycycline for 48 h. Total RNA was then isolated using a Quick RNA Isolation Mini Kit (Zymo Research). 1 μg of total RNA was reverse transcribed using iScript Reverse Transcriptase Supermix (BioRad). The relative levels of Firefly and Renilla luciferase transcripts in the sample were then quantified using Taqman qPCR. For each gene, the ratio of Firefly to Renilla luciferase in the case where the proximal UTR was fused to Firefly luciferase was set to 1.For each gene considered in this analysis , proximal and distal UTR sequences were extracted in such a way that they contained unique sequences only. This means that the distal UTRs of genes with tandem UTR models lacked the beginning of their sequence which is unique to the proximal UTR as illustrated in Fig. https://github.com/TaliaferroLab/FeatureReachR. This R package utilizes wilcoxon ranksum tests to compare length and GC contents of the three gene sets. Motif and five-mer enrichment significance is calculated with a Fisher’s exact test and corrected using the Benjamini & Hochberg method [http://cisbp-rna.ccbr.utoronto.ca/) [http://arescore.dkfz.de/arescore.pl).UTR sequence features of either positively or negatively correlated genes were always compared to the control gene set. Enrichment analyses were performed using a custom R package (FeatureReachR) publicly available here: g method . RBP binnto.ca/)  or RNA bnto.ca/) . AREScornto.ca/)  was utilAdditional file 1: Figure S1. (A) Read coverage plot of Elavl1 in mouse brain and liver tissues. Dots represent ψ values of 8 replicates. (B) PCA analysis of ψ values calculated from human tissues. Data was produced as part of the GTEx project. (C) As in B, but only using genes that have a tandem UTR APA structure. (D) As in B, but using only genes that have an ALE APA structure. (E) Comparison of ψ values from human brain and liver samples. Delta ψ values for genes with FDR values less than 0.01 are plotted. (F) Comparison of ψ values from human testis and liver samples. Delta ψ values for genes with FDR values less than 0.01 are plotted. (G) Read coverage plot of Dab2 in control human PBMCs and those treated with poly dI:dC. RNA from these cells was profiled using 3′ end sequencing. Dots represent ψ values calculated in each of 3 replicates. (H) Comparison of ψ values from 3′ end sequencing data as calculated by LABRAT (orange) and by counting aligned reads  (I) Comparison of APA quantifications produced by LABRAT (ψ) and QAPA (PPAU). (J) Benchmarking of APA software performance at a range of sequence read depths.Additional file 2: Figure S2. (A) Genes that repeatedly display differential APA isoform localization across repeated neuronal samples. Hierarchical clustering of ψ values from biochemically fractionated Drosophila DM-D17-C3 cells (B), HepG2 cells C), and K562 cells (D). (E-F) Simplex plots relating relative ψ values for genes between the cytosolic, membrane-associated, and insoluble fractions of DM-D17-C3 cells (E) and K562 cells (F). A dot that is equidistant from all three vertices had equal ψ values in each fraction while a dot that is closer to one vertex had a higher ψ value in that fraction relative to the other two fractions. (G-H) Comparison of ψ values in K562 (G) and DM-D17-C3 (H) cytosolic and membrane fractions for genes whose ψ value was significantly different between these compartments (FDR < 0.01).Additional file 3: Figure S3. (A) α values for each RBP knockdown in K562 cells were calculated using tandem UTR and ALE genes independently. These were then plotted and correlated. Each dot in this plot represents one RBP knockdown experiment. (B) Binomial p values for overlaps between genes whose APA was sensitive to RBP knockdown and genes whose 3′ UTRs were bound by an RBP in eCLIP experiments. Data taken from ENCODE HepG2 experiments. (C) As in B, but using data from ENCODE K562 experiments. (D) As in Fig. p < 0.05). To assess whether this was more than the expected number of significant RBPs, relationships between RBPs and their lists of APA and eCLIP targets were shuffled 1000 times, and the analysis was repeated after each shuffle to create a null distribution (blue).  As in Figs. S3D and 4E, but instead of considering eCLIP binding events only in the 3′ UTRs of genes, eCLIP binding events throughout gene bodies were considered.Additional file 4: Figure S4. (A-B) Histogram of gene-wise correlations between changes in ψ and changes in gene expression (ρ) derived from TCGA tumor and matched normal samples for tandem UTR (A) genes and ALE (B) genes. (C-D) Histogram of gene-wise correlations between changes in ψ and changes in gene expression (ρ) derived from ENCODE RBP knockdown and control samples for tandem UTR (C) genes and ALE (D) genes. (E) Binned scatter plot comparing changes in ψ and changes in gene expression for genes with negative ρ values (blue), positive ρ values (purple) and control genes (gray). (F) Enrichment of 5mers in the distal UTRs of negatively correlated genes compared to the distal UTRs of control genes. (G) Enrichment of 5mers in the distal UTRs of positively correlated genes compared to the distal UTRs of control genes.Additional file 5: Table S1. Delta ψ values (defined as RBP knockdown - control knockdown) for all RBP knockdowns in the ENCODE HepG2 data. Only genes with significant FDR values (less than 0.05) are shown. Delta ψ for genes that did not meet this threshold are indicated as NA.Additional file 6: Table S2. Delta ψ values (defined as RBP knockdown - control knockdown) for all RBP knockdowns in the ENCODE K562 data. Only genes with significant FDR values (less than 0.05) are shown. Delta ψ for genes that did not meet this threshold are indicated as NA.Additional file 7: Table S3. Correlation of expression changes and APA changes in TCGA and ENCODE data. A positive correlation indicates that an increase in gene expression was associated with an increase in ψ. Put another way, a positive correlation indicates that increased gene expression was associated with increased distal polyA site usage, while a negative correlation indicates that increased gene expression was associated with increased proximal polyA site usage. Spearman correlation coefficients were calculated across all sample pairs  in which the gene was expressed (TPM > 5). If a gene was not expressed in any sample pair, the correlation is noted as NA.


S = 3/2 unit corresponding to an existingmolecule, and the scaling to larger spin systems is quantitativelyanalyzed.Weshow that molecular nanomagnets have a potential advantage inthe crucial rush toward quantum computers. Indeed, the sizable numberof accessible low-energy states of these systems can be exploitedto define qubits with embedded quantum error correction. We derivethe scheme to achieve this crucial objective and the correspondingsequence of microwave/radiofrequency pulses needed for the error correctionprocedure. The effectiveness of our approach is shown already witha minimal  In standard approaches these extra-statesare obtained by encoding a logical qubit into many physical units.18 However, this makes the practical implementation of QEC and thecorresponding quantum computation extremely difficult, because nonlocalquantum gates on a large set of physically distinct objects are needed.10 A possible way to overcome this hurdle is byemploying a single multilevel quantum object to encode a logical qubit.24The route toward quantum computershas seen an astonishing boost in the past few years,30 thus paving the way to data storage at the single-molecule level.Magnetic molecules were also largely investigated as promising platformsfor quantum computation: interesting complexes were designed to meetspecific schemes39 and were chemically optimized to reach very long coherence times.44 As far as QEC is concerned, one could think of mapping 2n molecular levels to n qubits48 and applying standard error-correction codes for independent qubits.However, usually this does not work because real hardware errors inthese molecular systems do not typically translate into single-qubiterrors, thus making standard codes ineffective.49In this respect, molecular nanomagnets represent the idealplatform,offering many accessible (electronic and nuclear) spin states whichcould be used to encode a protected qubit. Chemical engineering enabledthe realization of molecular systems targeted for specific applications.For instance, careful tailoring of the ligand cage surrounding rare-earthions enabled the synthesis of bistable single-ion magnets showinghigh-temperature magnetic hysteresis,d levels of a molecular nanomagnet (qudit encoding), endowed with a QEC scheme to protect itagainst the most harmfulerrors occurring in molecular qubits, namely, pure dephasing. To thisaim, rather than resorting to codes based on abstract generic errormodels,50 we derivea code which is specific for the class of systems we are consideringand hence gives substantially better performance. We introduce error-protectedstates in such a molecular qudit and design the full sequence of magneticpulses actually realizing the QEC for generic spin systems. Alreadyexisting monomers and dimers can be used to implement our proposal,with an electronic or nuclear spin S spin S weakly coupledto a spin 1/2. In the former case μ is the nuclear magneton(μN), while in the latter μ ≡ μB. The first two terms in B along z; the thirdterm is the single-ion anisotropy of the qudit , and the last term representsa weak exchange or hyperfine ancilla–qudit coupling Γ. Because Γx,y is much smaller than the difference ofqudit and ancilla excitation energies, the eigenstates are simpletensor products of the eigenstates of Sz (|m⟩) and σzA  is the density operatorof the qudit.  Conversely, spin relaxationis usually very slow at low temperature in these systems, with electronicrelaxation times reaching ∼102 ms.54 Hence, our aim is finding how to use qudit states to correct forthe unwanted decoherence due to Supporting Information, for small t/T2 itis possible to perform the perturbative expansion error operatorsThemost important error in molecular qubits is given by pure dephasing,t/T2 only low powers of Sz affect the dynamics. By consideringthe matrix elements of Szn wethus define protectedqubit states (code words) and a QEC procedure torecover the effects of pure dephasing (up to a given order in (t/T2)n). Inspired by binomial codes on bosonic systems,56 we introduce protected code words:L⟩(|1L⟩) includes only odd (even) k in the range . Note that for correctingdephasing up to order (t/T2)n (for integer n) oneneeds at least 2n levels (see the Supporting Information). Hence, using an integer spin Sint is admissible, but it does not lead to abetter performance as compared to Sint – 1/2. We thus focus here on half-integer spins. As we showin the Supporting Information, these codewords ensure that (i) Ek errors bring |0L⟩ and |1L⟩ to distinguishable  states and (ii) the coefficients α and β of a generic superpositionof the logical states (α|0L⟩ + β|1L⟩) are preserved. Therefore, errors are detectable,for (i), and correctable, for (ii).57 We quantify the ideal performance of this code by computing the fidelity58 of the corrected state ρC(t), 0⟩ = α|0L⟩ + β|1L⟩. The final error (probabilityof finding the system in the wrong state) S. A comparison with a spin 1/2 without QEC(dashed line), shows a remarkable reduction of the error S.This shows that at short 59 and remarkable electronic coherence. Asfar as electronic spins (ii) are concerned, Gd complexes  can be exploitedas an additional resource. A simpler implementation is given by aCrYb electronic spin dimer,47 where theeffective Yb3+ spin doublet provides the ancilla and Cr3+ the 4-level qudit.Simple candidates to implementthe scheme are molecular complexesconsisting of (i) a single magnetic ion coupled to a magnetic nucleusor of (ii) pairs of electronic spins linked by exchange interactions.Several compounds belonging to class (i) exist, with nuclear spinsranging from 3/2 to 7/2d in ref ) can proPulse Sequence. The implementation of this QECcode requires translating the abstract operations above into preciseexperimental steps. To achieve this, we design for a generic spin S a complete sequence of resonant microwave/radiofrequencypulses inducing Δm = ±1 transitions. S = 3/2 case, while the general procedureis detailed in the Supporting Information. Starting with the qudit in a state |ψ(0)⟩ = α|−1/2⟩+ β|−3/2⟩, encoding in the protectedstates is achieved using the four pulses in the left part of the figure.After free evolution of the system (memory time),the error detection procedure is applied. With S = 3/2 only Sz errors can be corrected, and hence, we need to distinguish betweenthe no-error and Szerror cases. To detect the possible error,we excite the ancilla only if no Sz error has occurred, bringing the code words |0L/1L⟩ to error words ∼Sz|0L/1L⟩. Thisis achieved by first applying pulses that bring the correct state|ψ(0)⟩ to α|3/2⟩ + β|−1/2⟩,and the state corresponding to an error to α|1/2⟩ + β|−3/2⟩.Then, two simultaneous π pulses are employed to excite the ancillaonly if the qudit is in |m = −1/2, 3/2⟩(thanks to the ancilla-qudit coupling Γ). A subsequent measurementof the ancilla projects either into the no error . Detailson the general procedure for larger spin S are givenin the Supporting Information.Larger spin qudits allow us to correct more Physical Implementation. General requirementsto implement our scheme are the following: (i) Γx,y much smaller than the differencebetween excitation energies of qudit (Δm =±1) and ancilla (for nondemolition readout); (ii) significantΓz, to enable selective excitationof the ancilla depending on the |m⟩ stateof the qudit and hence error detection. These conditions can be fulfilledby applying a sizable magnetic field. The latter can lead to |m⟩|4)2[Cu(mnt)2] complex reportedin ref  excitations used for error detection(see the Supporting Information), whileit is kept in its ground state for the rest of the time. For thisreason, we can neglect spin relaxation on the electronic ancilla.54din ref , consistTQEC required to implementthe QEC code, making pure dephasing effective also during this step,as well as imperfect pulses (due to finite bandwidth of the Gaussianpulses) yielding gate errors. It is worth noting that our QEC schemeworks well even in these realistic conditions, as shown in optimal working point T̃ as the value of T corresponding to the maximum . T2 and even small S, we obtain a very large T̃/τ, roughly representing the number of operationswhich can be performed before we need to correct.S (dueto improved QEC) and T2. S (see the Supporting Information), thus marking a trade-off between the increase in the number ofcorrectable errors (number of powers of Sz whose effect can be corrected) and the effect ofdephasing during the correction procedure. Remarkably, our procedureyields large error reductions  Itexploits the many levels in the Hilbert space as a resource, ratherthan considering them as a leakage source only. (ii) It largely reduceshardware overhead when building up a processor. (iii) It makes logicaloperations  easier to realize. In addition,as compared to other multilevel codes which are based on generic errormodels,21 our scheme is targeted to the major errorsource in the real system. These reasons make our route promisingfor the realization of a scalable quantum processor.64In summary, we have shown that molecular nanomagnets, thankstotheir spectrum characterized by many accessible levels, can be usedto encode robust error-corrected qubits in single molecules. The single-objectnature of the logical units yields several advantages, compared tostandard multiqubit platforms:D to resolve all transitionsby magnetic pulses. It could also be extended to a wider class ofmolecular systems, by exploiting the flexibility in the level structureto achieve stronger qubit protection. For instance, molecules withcompeting interactions are characterized by many low-spin low-energymultiplets,72 thus enabling the increase of the number of levels without havinglarge Δm between the states (thus reducingdecoherence). Matrix elements between different multiplets can bechemically engineered by using ions with distinct g values, thus reducing the number of operations required by the code.As a result, more effective encodings could be found, involving potentialprotection against different classes of errors.We finally point out that the proposed scheme can be realizedusinga variety of magnetic molecules with large nuclear or electronic (effective)spin and a significant


The use of single-crystal substrates as templates for the epitaxial growth of single-crystal overlayers has been a primary principle of materials epitaxy for more than 70 years. Here we report our finding that, though counterintuitive, single-crystal 2D materials can be epitaxially grown on twinned crystals. By establishing a geometric principle to describe 2D materials alignment on high-index surfaces, we show that 2D material islands grown on the two sides of a twin boundary can be well aligned. To validate this prediction, wafer-scale Cu foils with abundant twin boundaries were synthesized, and on the surfaces of these polycrystalline Cu foils, we have successfully grown wafer-scale single-crystal graphene and hexagonal boron nitride films. In addition, to greatly increasing the availability of large area high-quality 2D single crystals, our discovery also extends the fundamental understanding of materials epitaxy. The heteroepitaxial growth of crystalline 2D materials is usually based on single-crystal substrates. Here, the authors demonstrate that single-crystal graphene and hexagonal boron nitride films can be successfully grown on wafer-scale copper foils with tailored twin boundaries. By judiciously choosing single crystal substrates with the appropriate surface texture, epitaxial growth of large area single-crystal graphene, hexagonal boron nitride (hBN), and other two-dimensional (2D) materials have recently been realized16. Despite significant progress in the synthesis of single-crystal substrates, such as Cu foils with different surface indices19, the preparation of single-crystal substrates is still generally expensive. Thermodynamically stable twin boundaries are common in face centered cubic (FCC) metals and are easily created during annealing treatments22. The presence of highly stable twin boundaries in Cu foils makes synthesizing high quality Cu single crystals challenging, and if possible, using polycrystalline substrates for 2D materials epitaxy would be highly beneficial.Wafer-scale single-crystal two-dimensional (2D) materials are highly desirable for the next generation of 2D material-based integrated electronics, optoelectronics and spintronicsHere we present a theoretical prediction and experimental demonstration of epitaxial growth of single-crystal 2D materials on tailored non-single-crystal substrates. We have successfully annealed polycrystalline Cu foils into various types of inch sized twinned Cu foils. Following the theoretical prediction, inch-sized 2D materials (including graphene and hBN) single crystals on selected twinned Cu foils have been synthesized. This study opens a rapid, scalable and robust route for single crystal 2D materials growth on tailored polycrystalline substrates.ψ, are the two independent variables that fully describe the twinned structure. In the ψ) are equivalent to Euler angles or surface indices for determining the orientation of a twinned crystal and their transformation. Such a twin boundary carries extremely low formation energy and can rarely be avoided during annealing of FCC metals22. The position of some typical Cu twins containing at least one low-index surface plane in the  map are presented in Supplementary Fig. The epitaxial growth of a 2D single crystal on a polycrystalline substrate requires that all 2D islands grown on two sides of a grain boundary are perfectly aligned, which is obviously impossible for an arbitrary grain boundary. Let’s consider the alignment of 2D material islands on the two sides of a 60° <111> twin boundary. The schematic diagram of a 60° <111> twin of a Cu foil with FCC structure is shown in Fig. 27. Previous studies reported that the alignment of 2D material  islands on a single-crystal Cu (1 1 1) surface is determined by the alignment of a zigzag edge of the 2D island with respect to a Cu <110> step edge29. However, an arbitrary FCC Cu surface may have many other types of step edges . Taking all these possible step edges into consideration makes it complicated to a priori determine the most favorable alignment of 2D material islands.To explore the alignment of 2D islands on twinned substrates, it is necessary to know the principle that governs the alignment of 2D materials on arbitrary surfaces. Supplementary Fig. 31. Taking graphene as an example, the interfacial formation energies of various graphene edge with respect to different metal step edge are calculated, see Supplementary Note To understand the mechanism of alignment of 2D material islands on an arbitrary Cu surface, we investigated the stability of the interfaces of various 2D material edges with different metal step edges33. For example, Au substrates are promising for the epitaxial growth of transition metal dichalcogenides (TMDCs)35, sharing the same epitaxial relationship as that of hBN on Cu surface. Due to the same 3-fold symmetry of TMDCs and hBN crystal lattices, the alignment TMDCs on a twinned Au substrate is the same as that of hBN on a twinned Cu substrate of same type and, thus, the synthesis of single-crystal TMDCs on twinned Au surfaces is possible.Here we selected the growth of two representative 2D materials, 3-fold symmetric hBN and 6-fold symmetric graphene, on twinned FCC Cu substrates as examples to demonstrate well-aligned 2D islands on both sides of a twin boundary. Including all possible twinned Cu foils, we calculated maps of misalignment angles between 2D material islands on the two sides of twin boundaries and the results are shown in Fig. 37 associated with a small twinned grain. If an indented Cu foil is bent along the transvers direction (TD)41, only one kind of twin structure is formed in the whole foil, as exhibited in Fig. Experimentally, a variety of twin structures can be obtained by subjecting raw Cu foils to deformation and annealing treatments. Figure In a similar manner, by changing the experimental parameters , a variety of twin structures can be obtained. For example, if an indented Cu foil was bent along a direction 45° to the TD direction, the whole foil showed another kind of twin structure (6 5 5)/(10 2 1), as shown in Fig. Figure 43. No etching lines were observed between aligned hBN domains in Supplementary Fig. The aligned domains on twinned Cu substrates eventually coalesce to form a thin film. To confirm this, we performed high-resolution TEM to characterize the merging area of domains at the atomic scale. The scanning TEM-annular dark-field (STEM-ADF) image and intensity profile of the merging area in Fig. 44 were adopted and we further confirmed the seamless coalescence of well-aligned graphene islands, nucleated on both sides of the Cu twin boundary in Supplementary Fig. 2 areas of the as-grown graphene/Cu sample was tested by low-energy electron diffraction (LEED) 51. Exchange-correlation functions were treated according to the generalized gradient approximation (GGA)52, and the interaction between valence electrons and ion cores was modeled by the projected augmented wave (PAW) methods53. Along the direction of the interface of a graphene edge attached to a Cu step edge, the lattice mismatch between the graphene edge and the step edge in the supercell was limited within 3.0%, and the graphene lattice was used to avoid extra strain on graphene. Along the direction perpendicular to the step edge, the distance between parallel step edges was maintained at a constant value of ~20 Å. In the out of plane direction, the vacuum space between neighboring images was kept at least 12 Å to avoid periodic imaging interactions. A force on each atom of less than 0.01 eV/Å with an energy convergence of 10−4 eV, was used as the criterion for structural relaxation. Computational details are discussed in the supplementary information.Calculations of the interfacial formation energy of graphene with different edges attached to Cu steps were performed within the framework of the DFT-D3 method2/Ar (30/500 sccm) ambient gas under atmospheric pressure. A quartz tube with a diameter of 80 mm was used to load the samples into the furnace. The Cu foil was bent to fit the quartz tube and annealed at 1010 °C for 1 h; a central hardness indent was created using a pyramid-shaped diamond hardness indenter under 300 g load and 10 s dwell time at the center of the Cu foil.In this study, we used commercial oxygen-free high conductivity (OFHC) Cu foils with a purity of 99.99 wt% from CHINALCO Shanghai Copper Company. The cold-rolled oxygen-free high purity polycrystalline Cu foils had a thickness of 46 µm and hardness of 132 HV (measured using a load of 50 g). X-ray diffraction measurements showed that the initial texture is composed of typical rolling components of S {123}<634> , Cu {112}<111> (16.6%), Brass {011}<211> (19.7%) and Goss {011}<001> (5.8%). The annealing treatments were conducted in a 100 mm-diameter furnace in H4  served as the B and N source. The growth was performed after completing the Cu annealing process to obtain a twinned Cu substrate. The gas ambient was H2/Ar (10/200 sccm) under 200 Pa pressure for 45 min at 1010 °C to grow a single crystal hBN film, while heating the precursor at 65 °C within 10 min using a water bath.hBN film was grown on a twinned Cu substrate by a chemical vapor deposition method. BNH4/H2/Ar (0.1/40/500 sccm) under atmospheric pressure and growth was carried out for 15 min at 1010 °C to obtain single crystal graphene film. Using a CH4 concentration of 185 ppm, it took approximately 15 min to fully cover the Cu surface with monolayer single-crystal graphene. To investigate the grain structure of graphene, the reaction was terminated before the Cu surface was completely covered by adjusting the experiment time from 1 to 15 min.Graphene film was grown on twinned Cu substrate by a chemical vapor deposition. Growth was performed after completing the annealing process to obtain twinned Cu. The gas ambient was CHRaman spectroscopy and mapping were performed using a He/Cd laser  excitation source at 1 mW power. The objective lens 50X was used and the spatial resolution was 1 μm. The accumulation time for each spectrum was 0.5 s for image scanning and 1 s for a single spectrum.2/Ar under atmospheric pressure. To visualize the etched areas, each sample was oxidized in air on a hot plate at 190 °C for 5 min. Oxygen passed through the etched regions to react with the underlying Cu substrate to form CuO while the Cu substrate under other areas remained unreacted.Etched hexagonal holes appeared on graphene after 10-min etching at 800 °C with 10/500 sccm HSEM and EBSD measurements were performed with a JEOL 7640/Oxford EBSD nano system. The working voltage was 10 kV.TEM was performed on twinned Cu structures and merging hBN domains. The twinned Cu was cleaned by electrochemical polishing, followed by conventional ion milling using a Gatan Model 691PIPS. HR-TEM images and the corresponding SAED patterns were obtained on an aberration-corrected TEM (FEI Titan3 G2) with an acceleration voltage of 300 kV. A double-tilt analytical holder was used. In the twin structure measurement, rotation angles α and β were 20.56° and −7.75°, respectively. After α and β rotations, the twin boundary was perpendicular to the sample holder, making the twin structure easier to observe.−10 Torr. The LEEM/μLEED measurement was performed on The LEEM III system  with operating pressure at 4 × 10−10 Torr.Low Energy Electron Diffraction (LEED) images were obtained from single-crystal graphene film on twinned Cu foil using a BDL600IR system from Omicron. A 1 cm × 1 cm section of the sample was cut and attached to a metal holder and introduced to a chamber under ultrahigh vacuum with operating pressure at 1 × 102/Si substrate by a wet transfer process using 4 wt% polymethyl methacrylate (PMMA) in ethyl lactate. The PMMA was spin-coated onto the graphene at 500 rpm for 5 s and 4000 rpm for 40 s and the samples were then heated on a hot-plate at 90 °C for 10 min. The samples were then treated in an oxygen plasma etching chamber  for 60 s to remove the hBN and graphene from the backside of the Cu foil. The treated samples were allowed to float on the surface of a Cu etching solution  for 4 h. The PMMA/hBN(Graphene)/Cu was transferred into another solution , allowed to stand for 30 min. and soaked in DI water for 30 min. The obtained PMMA/ hBN (Graphene) was then transferred onto the target substrate. The PMMA/ hBN(Graphene)/substrate was heated on a hot plate at 100 °C for 30 min, followed by soaking in acetone, alcohol, and finally in DI water.After CVD growth, hBN and graphene were transferred onto a 300-nm SiO2/Si substrate by wet transfer method. Electron beam evaporation and thermal evaporation methods were used to form electrical contacts , while oxygen plasma etching  was used to produce graphene ribbons. A series of FETs of different sizes  were produced for our research.Graphene field-effect transistors were fabricated by ultraviolet light lithography. Graphene films on Cu were transferred onto a 300 nm SiOIds-Vg) of the graphene FET was measured on a probe station with a Keithley 4200 semiconductor characterization system at room temperature under normal pressure. The widely-used Kim model based on nonlinear fitting was used to calculate the mobility:Ids is the current between the source and drain electrodes, Vds is the drain voltage with source grounded, Vg is the back-gate voltage, Rtotal is the resistance between the source and drain electrodes, Rcontact is the contact resistance of the source and drain, L is the graphene channel length ranging from 50 μm to 2 mm, W is the graphene channel width fixed at 10 μm, no is the carrier density at the Dirac point (residue carrier density), n is the carrier density induced by the back-gate bias away from the Dirac point, μ is the carrier mobility, e is the electron charge, VDirac is the gate voltage at the Dirac point, C is the dielectric capacitance of silicon oxide, ħ is Planck constant, and vF is the Fermi velocity of carriers in graphene, which is 1.0 × 108 cm/s. Three parameters, namely, the mobility (μ), residue carrier density (no), and contact resistance (Rcontact). were fitted. To improve fitting consistency, we measured more than seven graphene FET devices for each channel length.The transfer curve  and C2H4 (99.95% purity). The substrate is pretreated 50 μm-thick Cu foil (99.999% purity). CVD growth was performed at temperatures ranging from 700 degree to 1000 degree, with a pressure in the chamber ranging from 10−3 Pa to 25 Pa.In situ CVD growth experiments were performed inside the chamber of a modified commercial ESEM (FEI Quantum 200). After sample loading, the chamber was pumped out to around 10Supplementary Information


P > 0.10) but rather livestock farming system dependent. The ES rely more on the rearing management than on the breed type, and up to now, there are no specifications in Guadeloupe to differentiate management between breeds.The present study aims to assess (1) the ecosystem services (ES) provided by LFS and (2) the differential ES between local (Creole) and exotic breeds from pig, cattle and goat. The ES are defined as the benefits that humans derive from LFS. They were summarized in 12 ES indicators that cover services related to provisioning, ecological and socio-cultural aspects and territorial vitality. A total of 106 LFS units that covers the five agroecological zones of Guadeloupe were analysed. Functional typologies of LFS per species were created from surveys. The effect of breed on the ES indicators was tested. Results showed that the 40 pig LFS units were separated into 3 clusters that were differentiated in ES according to provisioning ES (cluster 1), cultural use and sale to the neighborhood (cluster 2) and pork self-consumption (cluster 3). The typology of the 57 farms with cattle distinguished 4 clusters with differences in ES provided in self-consumption (cluster1), ecological ES (cluster 2), socio-cultural ES for racing or draught oxen (cluster 3) and ES associated with territory vitality (cluster 4). The 66 goat LFS units were classified into 3 clusters different in ES concerning self-consumption (cluster 1), cultural aspects (cluster 2) and provisioning ES (cluster 3). Our study highlights that ES indicators are not breed dependent ( Another objective of this study was to investigate if the rearing of local breeds modifies the ES provided by LFS.Livestock farming systems (LFS) are at the centre of the debate on their ability to meet global challenges of food security, the limitation of greenhouse gases and fossil energy consumption and finally the preservation of biodiversity (Poore and Nemecek 2) and Grande-Terre (586 km2). The archipelago of Guadeloupe is characterized by five agroecological regions (AER) .All statistical analyses were carried out in the software R , while the variables “customers”, “subsidies” and “declared farmers” appeared more correlated to PC2 . The illustrative variable “number of exotic pigs” is correlated with these two PC . The PC3 and PC4 are more explained by the variable “neighbourhood” . From the HCA, pig farms have been classified into 3 clusters.Cluster 1: The first group was comprised of three intensive pig farming systems (between 631 and 835 pigs from synthetic line Duroc × Pietrain × large white × landrace or purebred large white). Farmers in this cluster aimed at producing 26 weaned piglets per sow per year. The indicators of ES provided by cluster 1 are summarized in Fig. Cluster 2: The second cluster was comprised of 23 farmers. The number of pigs was between 1 and 19 exotic, crossbred or Creole pigs. The main objectives of these farmers were (i) to produce pork for Christmas and other events during the year, (ii) to sell meat and/or animals to friends and to the neighbourhood and (iii) to produce meat for their own consumption. The ES indicators described for cluster 2 Fig.  are therCluster 3: In the third group (14 farmers), 79% of the farmers mainly reared Creole and/or crossbred pigs. Farmers were mainly oriented toward producing pork for their own consumption, and mainly for family events, such as Christmas. Only 14% of these farmers sold pork (1 to 2 pigs per year). This group is characterized by small-scale undeclared pig farms with no public subsidies. Most of the farmers (79%) reared other species . The pig housing system was close to the family house , either in small pens or tethered to a tree . Pigs were usually fed with leftovers, sugarcane, fruits , food crop residues and concentrates as a complement (between 50 and 100% of on-farm pig feed). The ES indicators described for cluster 4 Fig.  are therThe first two principal components (PC) explained 59% of the variability of the pig sub-database Fig. . The PC1r = 0.64, p < 0.001), “declared farmers”  and “upstream”  for PC1, and the variables “customers” , “upstream”  and “actives”  for the PC2. The PC3 is more correlated with the variable “animals sold”  and the PC4 with the variable “self-consumption” . The following HCA resulted in 4 main clusters . These two farmers reared on average one Creole cattle per year. The ES indicators studied from this group, shown in Fig. Cluster 1: The first group was comprised of only two farms. These two farms were characterized by a level of self-consumption significantly greater than the level of the other groups . These farmers were mostly in the formal sector . The majority of farmers in cluster 2 (59%) were also crop producers, mainly in sugarcane or banana production (from 1 to 16 ha). These crop products  and the resulted by-products  were used as animal feed. As shown in Fig. p < 0.001).Cluster 3: The third cluster was comprised of 12 farmers. These farms reared mainly crossbred cattle, ranging from 4 to 32 animals. All these farms were administratively declared. The grasslands ranged from 2 to 17 ha. Cattle were mainly tethered, and much of their feed came from the farm (73%). The ES provided by these LFS were summarized in Fig. p < 0.001). On the other hand, the values of ecological ES provided by the farms of cluster 4 were lower than in the other cattle farms, related to a lower level of cattle feed self-sufficiency  due to a lower level of grassland used for feeding . In addition, like in cluster 3, farmers of cluster 4 delivered higher socio-cultural ES than farmers from clusters 1 and 2, with animals used for cattle racing and draught oxen.Cluster 4: The last group was comprised of 11 farmers. These farmers were in the formal sector with large numbers of Creole and crossbred cattle . The ES assessment of cluster 4 showed that the ES associated with territory vitality were more important in this group than in other groups, with a level of upstream stakeholders and customers significantly greater than in the other groups : “declared farmers” (r = 0.75), “upstream” (r = 0.74), “subsidies” (r = 0.71) and “feed self-sufficiency” (r =  − 0.62). The second PC explained 15% of the total variance and was more correlated (p < 0.001) with the variables “subsidies” (r = 0.58), “declared farmers” (r = 0.53) and “neighbourhood” (r =  − 0.55). The variables “cultural uses” (r =  − 0.63) and “goat sold” (r = 0.53) contributed most to PC3 (p < 0.001) and the variables “goat sold” (r = 0.71) and “active” (r =  − 0.58) contributed most to PC4. The HCA discriminated goat farmers into 3 different groups . Only 7% of these LFS units were administratively declared for goat production. The majority of farmers in this group consumed the meat of their own goats (63% of farmers) and 46% of them slaughtered on the farm for their own personal consumption. Consequently, the ES provided by this group  in grassland areas of 2 ha on average. Goat self-consumption was more present in this group than in the other groups .Cluster 2: The second cluster gathered 15 farms, all of which were officially declared as goat farms in public administration. They all received specific funds from the European Union for rearing small ruminants. These farmers owned on average 30 goats, which was a lucrative activity. The farmers reared mainly Creole or crossbred goats (86%) and a few number of farms reared exotic breeds (21%). The production model was mainly intensive, and the farmers used grasslands of about 7.2 ha on average. Figure p < 0.001). As illustrated in Fig. p < 0.001). On the other hand, provisioning ES were well developed with a higher proportion of upstream and downstream stakeholders. As illustrated in Fig. Cluster 3: The last goat group is composed of 10 goat farmers mainly with Creole and crossbred goats (only one farmer reared exotic goats). The stocking rate was higher than in the other groups . The p-value is greater than 0.11 but the MATS p-value is lower than 0.05, indicating that there are significant differences in the ES indicators obtained by these two groups.In cattle, only Creole farms were compared with crossbred farms because there was only one farm with a herd containing more than 80% of exotic cattle. The WTS p-value greater than 0.5, the MANOVA indicates that there is no significant breed effect on the different ES provided by farms with Creole or crossbred goats.In goats, only Creole farms were compared with crossbred farms because there were only 3 farms with a herd containing more than 80% of exotic goats. With WTS and MATS Livestock practices directly generate ecosystem services, defined as the benefits that humans derive from ecosystems Leemans . MethodsIn this paper, we focused only on three species  reared in LFS in Guadeloupe because Creole breeds are well characterized in these species  or mixed crop-livestock systems suggest that the intensity of combining livestock species or crop-livestock is low in the majority of LFS studied. As observed by Stark et al. , mixed sThe following sections discuss the ES provided by LFS within species.Our study showed that the ES provided by pig LFS differed significantly between the 3 clusters identified, which could be attributed to the different purposes of farmers in rearing pigs. Farmers in the first cluster followed the intensive production rules of the European Union . The provisioning ES was consequently strong while the values of feed self-sufficiency level, which summarizes the pressure on the environment and the neighbourhood acceptance, were low. Similarly, Ryschawy et al.  reportedIn our study, cattle farms had common ecological ES related to management in grass-based systems either in grasslands or in small-scale rotational grazing. In Guadeloupe, rearing tethered animals and using rotational management are very frequent in cattle farmers. Boval et al.  showed hThe levels of ES provided by cattle LFS varied among the 4 clusters identified, mainly in ES related to socio-cultural services and territorial vitality. The socio-cultural services provided by cattle are mostly cart drawn by oxen and draught oxen, mainly from two clusters of farmers. The socio-cultural ES was low in the other clusters either because the cultural acceptance of the farms was moderate, as the neighbourhood complained or because the few cattle reared were for self-consumption. In the present study, cattle were the only species for which live animals were used in the cultural service (cart race). When investigating the ES provided by livestock in some European regions, Ryschawy et al.  showed tAs observed in this study, the highest values in provisioning services were associated with the largest farms. This is consistent with previous studies , rearing goats also took a fundamental dimension as a means of survival, as “no other income goes into the household”. In the second cluster, goats were found to be culturally important. The animals were often slaughtered for Catholic holidays  or sold or sacrificed for Hindu ceremonies. Alexandre et al.  showed tIn contrast to our expectations, Creole pig and Creole goat breeds did not provide specific ES compared to exotic and crossbred livestock. This result confirms the low correlations found between the illustrative variables  and the principal components in the PCA analysis. These results could be explained by the lack of additional or differential value given to Creole breeds. In general, ES provided by specific breeds are connected to labelled products allowing the promotion of specific characteristics of the breed in the marketing system (Bernués and Martin-Collado The present study demonstrates that the ecosystem services provided by livestock farming systems in the tropical conditions of Guadeloupe are multiple. Our study, by assessing and comparing ecosystem services provided by pig, cattle and goat systems, covers the larger part of the meat sectors in Guadeloupe. We found differences in services provided between species. We highlight that ES indicators are not breed dependent but rather livestock farming system dependent because ES provided by livestock rely more on the rearing management than on the breed type. Further studies involving assessment of ES at the territorial level combining farmers and other stakeholders should provide information to improve ES provided by livestock in Guadeloupe. Such studies could facilitate collective initiatives involving political decision-makers to develop a specific niche market that promotes the recognized qualities of Creole breeds, and help their conservation.


Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species.Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GINs). Some of the most promising alternatives are based on the immune response of the host, such as the selection of genetically resistant breeds or the use of vaccines against these parasites. Given the limited information available on the immune response against GINs in goats, this study investigated the local immune response of goat kids of an indigenous Canary Islands breed (Majorera breed) experimentally infected with For this purpose, the relationship between different parasitological  and immunological parameters at the local level  was analyzed at early (1 week post-infection [wpi]) and late (8 wpi) stages of infection.T. circumcincta infective larvae (L3) generated a complex immune response that could be defined as Th2 type, characterized by increased infiltration in abomasal tissues of several effector cells as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. Cellular responses were evidenced from 1 wpi onward, showing an increase in antigen-presenting cells and various lymphocyte subsets in the gastric mucosa.Primary infection of goat kids with +, CD4+, CD8+, γδ+, CD45R+, IgA+, and IgG+), as well as in the evolution of the relative cytokine gene expression. From a functional point of view, negative associations were observed between the number of most of the immune cells  and parameters that could be related to the fecundity of worms, a phenomenon that was especially evident when the number of IgG and CD45R cells or the specific IgA levels of the gastric mucus were compared with parasitological parameters such as the female worm length or fecal egg counts at 8 wpi.The complexity of the host response was evidenced by statistically significant changes in the number of all these subpopulations (MHCII Teladorsagia circumcincta stands out for its wide distribution and pathogenicity ), with a mean length of 3.14 ± 0.2 mm. After digestion of the gastric mucosa, 77 ± 33.4 larvae/g mucosa were detected.r) showed a nonsignificant negative association between the number of worms in the gastric content and their length. A statistically significant positive association between length and number of intrauterine eggs was shown (r(3) = 0.900, P = 0.037).In group 2, slaughtered at 8 wpi, the mean (± SEM) of total adult worm counts was 2039 ± 480.7. Female worms  showed a mean length (± SEM) of 8.32 mm ± 0.6 mm and a mean number of intrauterine eggs (± SEM) of 9.03 ± 2.1 eggs/worm. The number of larvae in the mucosa (mean ± SEM) was 6.6 ± 1.6 larvae/g. Correlation analysis by Spearman's test  showing a mean FEC of 370 EPG ± 80.5 at the end of the experiment Fig. .Fig. 1EgT. circumcincta developed a strong reaction against antigenic fractions with molecular weights of approximately 22, 39, 50, 84 and > 120 kilodaltons (kDa)  = −0.975, P = 0.005).At the local level, the presence of specific IgA in the gastric mucus was much more evident in infected animals in which worms had reached the adult stage group 2) compared to group slaughtered at 1 wpi (group 1) and the uninfected control group , while animals from group 1 and uninfected controls showed a poor specific response  =  −0.894, P = 0.041). No statistical associations were observed between the other two effector cell populations (eosinophils and mast cells) and any of the parasitological variables studied.Infected groups 1 and 2 showed a clearly higher mean number of eosinophils infiltrated in the gastric mucosa than the uninfected control group, although with no statistical significance. The globule leukocytes and mast cells counts were also higher in the gastric mucosal from animals of the infected groups, but similarly these differences were not statistically significant Fig. . In the +, CD4+, MHCII+, and IgG+ cells in group 1, and γδ+, WC1+. and IgG+ in group 2 , all subpopulations of cells studied showed higher mean counts than in the control group. These differences were significant when comparing WC1p 2 Fig. .Fig. 5Le+ cells and immature worm burden at 1 wpi (group 1). Female worm counts at 8 wpi (group 2) were also positively associated with CD4+, γδ+ and IgG+ cells. On the contrary, negative correlations were detected between CD4+, γδ+, IgG+, IgA+ or CD45R+ cells and worm length (at both 1 and 8 wpi). At 8 wpi, these subpopulations of lymphocytes  also showed a negative association with FEC. However, all these negative correlations were not significant except when the relationship between FEC and IgG+ cells (r(3) = −0.875, P = 0.038) or FEC and CDR45R+ (r(3) = −0.900, P = 0.004) cells were analyzed at 8 wpi (group 2).Correlation analysis between parasitological data and immune cell counts in the abomasal wall showed positive associations between IgAAll experimental groups showed detectable gene transcription levels of IL-2, IL-4, IL-10, IL-17, and INF-γ in both lymph nodes and gastric mucosa Fig. . AlthougT. circumcincta in Canarian goats [T. circumcincta [Despite the homogeneity of the animals included in the different experimental groups in terms of age, sex, breed, weight, origin or feeding, the different parasitological parameters analyzed in the present study showed high variability, as observed in other experimental infections with an goats , 27, 28.umcincta –31. Thisumcincta , which wT. circumcincta [Similarly, the prepatency period in our study was approximately 25 days, which seems to indicate a slight delay in larval development compared to sheep, which has also been observed in other trials in goats using other isolates of umcincta , a phenoT. circumcincta infections is the reduction in FEC, mainly linked to decreased female fecundity rather than number of parasites [T. circumcincta in goats. Fecundity was also assessed by the intrauterine egg counts, which showed a logical positive correlation with length of these worms, a feature found in other studies for this parasitic species [T. circumcincta fecundity in this ruminant species.In sheep, among the mechanism associated with resistance to arasites . In our  species . NegativT. circumcincta has also been observed in sheep infected with this parasite [The relevant function of IgA in resistance against parasite –37. In tparasite . In the T. circumcincta immunoglobulins (IgA and IgG isotypes) at the local level. This significant enhancement was accompanied by other findings associated with Th2 responses, such as eosinophil infiltration of the mucosa and hyperplasia of mast cells and globular leukocytes [T. circumcincta [Haemonchus contortus [The humoral response observed in the gastric mucus corresponds to a Th2 immune response, which is considered a common feature in GIN infections. Th2 response was more evident in the infected group at 8 wpi (group 2). This group showed a significant increase in the level of anti-ukocytes . Althougumcincta  or Haemoontortus  in sheepT. circumcincta-infected goats, reaching higher counts at 8 wpi (group 2), but were also observed in the early stages of infection , a finding that could be associated with innate defensive mechanisms [H. contortus [The enhancement in the gastric mucosa of other effector cells, such as globule leukocytes and mast cells, has also been associated with natural resistance mechanisms against GINs . Both cechanisms . Eosinopontortus , 44. Howontortus .T. circumcincta, similar to that found in goats infected with other gastric nematodes, such as H. contortus [+) and CD4+ lymphocytes and worm burden, a finding that has been linked to the resistance of sheep against GINs [+, γδ+, IgA+, IgG+, or CD45R+ and fecundity  at 8 wpi, as observed when the local humoral immune response was analyzed. The presence of a significant CD8+ lymphocyte infiltrate observed here was also previously detected in goats and sheep infected with H. contortus from 7 to 10 days pi. However, similarly to what occurred in our study, no relationship could be established with the parasitological data [+ lymphocytes and their WC1+ subpopulation in the gastric mucosa from the first week of infection was also evident here. These cells constitute an important percentage of lymphocytes in goats, especially in young animals [H. contortus [H. contortus, a phenomenon associated, as in the current trial, to the local humoral response developed by specific IgA antibodies [T. colubriformis, where no significant defense mechanisms attributed to γδ+ cells could be identified [The present results also showed a rapid recruitment of lymphocyte subpopulations in the abomasal mucosa of goats primarily infected with ontortus . This finst GINs , 48. Thecal data , 46. The animals . As has tibodies  as discuentified . This fientified . This cyentified .H. contortus or Ostertagia ostertagi infection in goats and calves, respectively, could be contradictory if the inhibitory effect of INF-γ on Th2 responses is taken into account [When the experimental infection progressed, although a humoral response could be detected and some effector cell subpopulations were increased, the gene expression of mediators of the immune response was even less evident than during the first week of infection. Thus, only a slight increase in the relative expression of INF-γ in the gastric mucosa of group 2 compared to the control group was observed. This finding, which has also been reported in the course of  account . Such ob account –57, or e account .T. circumcincta L3 generates a complex immune response that could be defined as Th2 type, characterized by an increased abomasal infiltration of several effector cells  as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. These cellular responses were evidenced from 1 wpi onward, showing an increase in antigen-presenting cells (MHCII+) and various lymphocyte subsets  in the gastric mucosa. The complexity of the responses developed is evidenced by the statistically significant changes in the numbers of all these subpopulations, as well as in the evolution of the relative cytokine gene expression observed in the gastric mucosa and abomasal lymph nodes at both 1 wpi and 8 wpi. From a functional point of view, negative associations were observed between the number of most of these cells  and parameters that could be related to the fecundity , a phenomenon that was especially evident when the number of IgG or CD45R cells or the specific IgA levels of the mucus were compared with parasitological parameters such as the length of female worms or FEC at 8 wpi. Although more studies would be necessary, this information should be taken into account to evaluate alternative control strategies against T. circumcincta based on the host immune response, such as selection programs for resistant goat breeds or vaccination protocols against this nematode species.In conclusion, the primary infection of goat kids with