dataesr/scientific-paragraphs-categorization huggingface dataset:
2 , step 6). Ibarra-Soria and colleagues provided high-quality data, with over 138 million paired-end fragments from 3 C57BL/6 J adult male OMs (referred to as male_IS2014)  [26]. During the setup of the pipeline on these data, we considered that the precision of 3′ end annotation cannot exceed 100 nt for the following reasons: the read size is 75 nt, the cleavage sites at a single polyA site are observed in a 24-nt window  [60] , and a sequencing depth of 200 reads/kb is sufficient to reach a 100-nt resolution in IsoSCM transcript assembly with a 90% true positive rate  [41]. In the data set produced by Ibarra-Soria and colleagues, only 59. 4% of the Olfr genes exceed the sequencing depth of 200 counts/kb (in the CDS) for which a 100-nt resolution is achieved. Indeed, Olfr genes are expressed in a monogenic and monoallelic manner in OSNs and therefore most Olfr mRNAs show low abundance in whole olfactory mucosa extracts, limiting the precision of 3′ end identification for Olfr genes (see below).
We considered further the 3′ ends which were matched with a canonical PAS (AAUAAA or AUUAAA) situated in a [-100;+ 100] window. Overall, in our analysis, 67. 4% of the annotated 3′ ends are matched with a canonical polyA signal. Thus, we definitely used a precision window of 100 nt. From the 1181 Olfr genes investigated in adult male olfactory mucosa (male_IS2014 dataset), we were able to annotate 715 Olfr genes (61. 4% of the 1165 Olfr genes expressed, i. e. with at least 1 read in the CDS) with the IsoSCM pipeline (Additional file 3: Table  S2 ) and 908 Olfr genes with Cufflinks. For these annotated Olfr genes, our pipeline was able to detect 1547 3'UTRs with IsoSCM vs. 946 3'UTRs with Cufflinks. Furthermore, the mean number of 3′ ends per Olfr is 1. 03 and 1. 04 in the Cufflinksbased analysis by Shum and colleagues (File 010 Additional file 3: Table  S2  in  [27] ) and by ourselves (male_ IS2014 dataset), respectively, whereas it reaches 2.
First, we investigated the precise cleavage sites by RNA Ligation mediated PolyAdenylation Test (RL-PAT), an RT-PCR-based strategy. We confirmed the use of 5 out of the 8 polyA sites using this technique (Fig. 3f ; Table  1 ). Unfortunately, the 3 last sites could not be validated using this method, due to technical limitations: the presence of upstream repeat sequences made it impossible to design specific primers for testing Olfr1507 3'XL, Olfr15 3'L and 3'M1. Next, we demonstrated by northern blot the existence of alternative 3'UTR isoforms for both pilot genes using probes designed for the sequences either upstream (CDS probe) or downstream (3'M probe) of the proximal 3′ ends (Fig. 3g-h ). The Olfr1507 3′S isoform appears as Fig. 2  Computational pipeline for the characterization and analysis of Olfr 3'UTR isoforms from RNA-Seq datasets.
The pipeline is divided into six steps: Step 1: RNA-Seq reads are mapped using STAR, and reference genome and annotation files. Step 2: A mask file in bed format, restricted to the Olfr loci in mouse, is generated. Step 3: The mask genome alignment output is obtained. Step 4: Full known and novel transcripts are reconstructed with IsoSCM or Cufflinks. Step 5: Reconstructed transcripts are characterized and analyzed in terms of annotation, 3'UTR isoforms identification, identification of predictive PASs at 3′ ends, merging (merge of 3'UTR isoforms from the same gene when 3′ ends are distant from less than 100 nt) and 3′ intron detection. Step 6: The relative abundances of the multiple 3'UTRs generated for the same Olfr gene are assessed by RNA-Seq quantification. Rectangles: input files or output files.
3h ). The 3'L isoform of Olfr15, not detected using northern blot, is most probably expressed at a very low level (below the sensitivity of this technique; Fig. 3h ). The existence of an XS 3'UTR for Olfr1507 (described by Ibarra-Soria's study and in the Ensembl database; Fig. 1a ) was confirmed by RL-PAT, and this isoform is probably generated using a non-canonical PAS (Table  1 ). However, this 3'XS isoform is probably expressed at a very low level (below northern blot sensitivity -Fig. 3g ). Very importantly, northern blot confirmed the relative abundances of the alternative 3'UTR isoforms determined using RNA-Seq quantification (Fig. 3d ). In conclusion, our experimental investigations performed on these two pilot genes confirmed the data obtained in silico using our IsoSCM pipeline, from both the qualitative (repertoire of 3'UTRs) and quantitative (relative abundances of alternative isoforms) points of view.
Characterization of the full repertoire of detectable Olfr 3'UTRs using our IsoSCM pipeline As mentioned above, our IsoSCM pipeline allowed the characterization of 1547 3'UTR isoforms, generated from the 715 Olfr genes annotated from the male_ IS2014 dataset. As for our 2 pilot genes, the great majority of the annotated Olfr genes (77. 5%) show multiple 3'UTRs due to alternative polyadenylation, 44. 8% with 2 isoforms, 27. 4% with 3 isoforms and 5. 3% with 4 or 5 isoforms (Fig. 4a ). The 3' ends distant of less than 100 nt were merged as a single polyA site. b For each putative polyA site, predicted canonical PAS matching in a [-100;+ 100] window from the RNA-Seq-deduced 3′ end are indicated. c Note that for Olfr1507 3'XL isoform, the closest canonical PAS is slightly outside the 100-nt precision window.
This statistical analysis shows that our pipeline can discriminate between single 3'UTRs and up to 3 multiple isoforms due to APA, independently of the expression level of the Olfr genes A B C Fig. 4  A large majority of Olfr mRNAs shows multiple 3'UTR isoforms produced through alternative polyadenylation. The whole male_IS2104 dataset was analyzed using our IsoSCM pipeline. a Distribution of numbers of 3'UTR isoforms per Olfr. b Distribution of length for 3'UTR groups; sUTR = single 3'UTR for Olfr genes without APA (purple); pUTR = proximal 3'UTR isoform (dark green) and dUTR = distal 3'UTR isoform(s) (light green) for Olfr genes with APA. One-way Kruskal-Wallis test (Chi square = 430. 62, p < 0. 0001, df = 2), followed by Nemenyi test (* p < 0. 05, *** p < 0. 001). c Distribution of Olfr genes in 4 distinct quantitative 3'UTR isoform profiles.
Thus 29% of annotated Olfr genes produce several 3'UTR isoforms of quantitative importance (P3 or P4 profiles), as for Olfr15 (Figs. 4c  and 3d  and h ). In 71% of annotated Olfr genes an isoform represent more than 80% of the isoform either because it is a unique (P1 profile) or a major (P2 profile) isoform, as for the Olfr1507 pilot gene (Figs. 4c  and 3d  and g ). To characterize the contribution of alternative splicing to the 3′ alternative isoforms of Olfr mRNAs, we identified the introns included in this new 3'UTR repertoire. It turned out that only 13 Olfr genes display an obligatory or optional intron in their 3'UTRs with a depth > 1 (male_ IS2014 dataset, Additional file 6: Table  S3 ). As an example, the 3'M isoform of Olfr1508 is subject to an optional intron excision detected by STAR (Fig.
7). Comparison of 3'UTR isoforms annotated from 4 datasets reveals robust 3'UTR isoforms in both sexes, and no sexspecific 3'UTRs All the above analyses were done on the RNA-Seq dataset from 3 adult male OM published by Ibarra-Soria and colleagues in 2014 (male_IS2014)  [26]. In the same paper, a second dataset was obtained from 3 adult female OM (referred to as female_IS2014). An independent experiment with both adult male (male_IS2017) and adult female (female_IS2017) datasets was recently published by the same authors  [40]. We took advantage of the availability of these 4 datasets produced by the same laboratory to evaluate the robustness of our IsoSCM pipeline and to compare the Olfr 3'UTR repertoires between females and males.
Global comparative analysis of the annotations from the 4 datasets We processed these data with our IsoSCM pipeline to annotate the Olfr 3'UTRs. While the absolute numbers of annotated Olfr genes and 3'UTR isoforms obtained in the 4 datasets differ (715, 834, 625 or 743 annotated Olfr genes and 1547, 1810, 1344 or 1565 3'UTR isoforms for the male_IS2014, male_IS2017, female_IS2014 or fe-male_IS2017 datasets, respectively), these repertoires display a large overlap from one sample to another (Additional file 8: Figure  S5A-D ). As was the case for the male_IS2014 dataset, the detection of multiple (up to 3) 3'UTR isoforms did not depend on the expression level of the Olfr genes in the 3 other datasets (Additional file 5: Figure  S3 ). We however observed a differential expression of the Olfr genes between the samples from the 2 independent experiments (Additional file 8: Figure  S5E-H ).
Whereas male and female samples from the same experiment had similar expression levels, samples from the same sex from different experiments show significantly different expression levels, with an increased normalized expression in the 2017 experiment as compared to the 2014 experiment. This was not surprising because these 2 experiments are not biological replicates; they differ in animal housing conditions, sample preparation (stranded In addition, because the uncertainty regarding the position of polyadenylation sites reaches 200 nt between 2 annotations, we considered 3'UTR isoforms as "conserved" between 2 datasets when the length difference between the corresponding 3'UTRs annotated in the 2 datasets is less than 200 nt. According to this rule, 483 s/ pUTRs (of 639 Olfr genes annotated in both male datasets) and 247 dUTR1s (of 561 Olfr genes with an annotated dUTR1 in at least one male dataset, and 446 in both male datasets) are conserved between the 2 male experiments (Additional file 3: Table  S2 ).
Since the coverage decreases along the 3′ regions, the most distal 3'UTRs (dUTR2, dUTR3 and dUTR4) show higher probability to be either missed or misplaced (due to reduced precision of the assembly). Accordingly, the length of same-rank UTRs is better conserved between the 2 male datasets for single or proximal 3'UTR isoforms (75. 6% "conserved" s/ pUTRs), than for the distal 3'UTR isoforms (55. 4% "conserved" dUTR1s; 51. 4% dUTR2s, 35. 7% dUTR3s). The rare Olfr genes showing dUTR4 isoforms were not the same in the 2 male datasets. Therefore, we focused on sUTR, pUTR and dUTR1 3'UTR isoforms when comparing 3'UTR isoforms produced by individual Olfr genes in the different datasets. The global analysis of these 4 datasets confirmed a substantial APA for Olfr mRNAs both in male and female (77.
5, 78. 4, 76. 6 and 76. 7% of the annotated Olfr genes in the male_IS2014, male_IS2017, female_IS2014 or female_IS2017 datasets; Fig. 7a ). As for the first dataset of male_IS2014, we observed a large diversity in the length of the 3'UTRs, with significantly different lengths between the sUTRs, pUTRs and dUTRs within each dataset. There was no difference between the 4 datasets in terms of 3'UTR length for either sUTRs or pUTRs (Fig. 7b ), or in their relative abundance profiles (Fig. 7c ). However, a significant difference in dUTR length was observed between the 2 experiments (2014 vs. 2017), although not between male and female datasets (Fig. 7b ). For all parameters examined between the 4 datasets, these global investigations thus did not reveal striking differences, except a length difference for dUTRs between the 2014 and 2017 experiments, probably due to the technical differences in these 2 experiments.
Comparative analysis of the annotations for individual Olfr genes common to the 4 datasets Five hundred and one Olfr genes with at least one annotated 3'UTR isoform were retrieved in all 4 datasets. Our 2 pilot genes, Olfr1507 and Olfr15, turned out to belong to this group of 501 common Olfr genes. Their detailed in silico analysis globally confirmed the robustness of our pipeline across independent datasets, in terms of alternative 3′ ends and length of the 3'UTRs, even though minor differences were observed (Additional file 9: Figure  S6 ; Additional file 10: Table  S4 ). The absence of detection of 2 distinct 3′ ends for the 3'M1/2 PAS of Olfr15 could be attributed to the presence of a stretch of canonical PAS (see above). A very rare 3'XL was detected only in female_IS2014 but in neither male nor in female_IS2017 datasets.
This discrepancy might be explained by the limit in sensitivity of our technique for isoforms expressed at a very low abundance. From a quantitative point of view, the relative abundance profiles of 3'UTR isoforms are not statistically different for these 2 genes across the 4 datasets (Fisher exact test Olfr1507 p = 1; Fisher exact test Olfr15 p = 0. 24; Additional file 9: Figure  S6 ). This robustness encouraged us to further compare the annotations of Olfr genes between the 4 datasets (Additional file 3: Table  S2 ). We first searched for isoforms that are experiment-specific i. e. not conserved in samesex comparisons (male_IS2014 vs. male_IS2017 and fe-male_IS2014 vs. female_IS2017) but conserved between males and females in both experimental series (male_ IS2014 vs. female_IS2014 and male_IS2017 vs.
female_ IS2017 comparisons). Among the 501 common Olfr genes across the 4 datasets, we identified 27 experimentspecific s/pUTRs. Focusing on the 324 Olfr genes with multiple 3'UTR isoforms in the 4 datasets, we found 21 experiment-specific dUTR1s. On the other hand, we identified putative sex-specific isoforms, i. e. conserved in same-sex comparison but not conserved between males and females in both experimental series. Only 4 s/pUTRs (for Olfr133, Olfr1107, Olfr618 and Olfr1226) and 5 dUTR1s (for Olfr476, Olfr796, Olfr1226, Olfr653 and Olfr11271) display such an apparent sex difference (Additional file 3: Table  S2 ). Thus, as far as we can conclude from this study of 501 Olfr genes, we identified only marginal possibilities of sex-associated APA events in the 3'UTRs of Olfr mRNAs, whereas some experiment-specific annotations of Olfr 3'UTRs may arise because of technical differences between the 2 sets of experiments.
By contrast, we annotated 310 s/pUTRs and 114 dUTR1s that are fully conserved in the 4 datasets analyzed (independently of the experiment series or the sex of the mice, Additional file 3: Table  S2 ). These annotations constitute a list of very robust 3'UTR isoforms for Olfr mRNAs. Finally, we found only 6 Olfr genes with introns in 3′ regions in the 4 datasets (Olfr1508, Olfr273, Olfr303, Olfr399, Olfr620, Olfr706), and no sex-specific splicing events (Additional file 6: Table  S3 ). Altogether, our data demonstrate that the new Olfr 3'UTR repertoire identified through our IsoSCM pipeline correspond to cognate 3'UTR ends expressed in the adult olfactory mucosa, and that the large majority of these in silico characterized 3'UTRs are robustly identified independently of the biological or technical variations introduced by the choice of the RNA-Seq datasets.
Longer Olfr 3'UTRs than previously proposed In a recent study based on the use of Cufflinks, Shum and colleagues reported that the Olfr mRNAs have exceptionally short 3'UTRs, with a median length of 773 nt  [27]. In the present study, we clearly disprove this view, thanks to the use of a more appropriate method for 3'UTR analyses. As compared to Cufflinks, IsoSCM not only allows much better detection of APA, but also improves the detection of full-length UTRs (e. g. by merging transcript fragments separated by small gaps)  [41]. We have established that the median length of single 3'UTRs is 1. 0-1. 2 kb (depending on the dataset considered). While the median length of proximal 3'UTRs ranges from 0. 6 to 0. 8 kb, the median length of the distal 3'UTRs reaches 1. 9 to 2. 2 kb in the adult OM.
Thus, the 3'UTR length of the Olfr genes falls in the range of the 3'UTR length reported for other mouse genes in various studies  [32, 62, 63]. Among the Olfr genes with multiple 3'UTR isoforms (in the male_IS2014 dataset), we noticed that the shortest isoform (pUTR) is generally the most highly expressed (true for 95% of Olfr genes with 2 or more 3'UTR isoforms; Additional file 3: Table  S2 ). Thirtyeight % of the Olfr genes with multiple 3'isoforms have distal isoforms of quantitative importance (i. e. belongs to the P3-P4 quantitative profiles vs. P2). The fact that pUTRs are significantly shorter than sUTRs argues for a specific proximal UTR population, distinct from single UTRs.
Comprehensiveness of our 3'UTR annotations In their original RNA-Seq analysis, Ibarra-Soria and colleagues  [26]  reported the annotation of 913 Olfr fulllength gene models. Focusing on the identification of 3′ ends, our pipeline allowed the accurate annotation of only 600-800 of Olfr genes, depending on the dataset (corresponding to 53. 8-71% of the expressed Olfr genes). Consequently, our description of the 3'UTR landscape for Olfr mRNAs is not comprehensive. The analyses of the 4 datasets presented here show many substantial variations in the number, length and/or relative abundance of 3'UTR isoforms for individual Olfr genes (Additional file 3: Table  S2 ; Additional file 9: Figure  S6  and Additional file 10: Table  S4  for the pilot genes), most probably for the following reasons. Even with the same genetic background (C57Bl6) and similar age of the 2 groups of adult males or females, unavoidable biological differences may arise in independent same-sex datasets.
Accordingly, we observed that the dUTRs are less conserved at a fixed rank between the 2 male datasets than sUTRs or pUTRs, and that the conservation decreases with the rank of the dUTRs (dUTR1 > dUTR2 > dUTR3 > dUTR4). Moreover, non-coding sequences show an increased frequency of repeat sequences as compared to CDSs, producing gaps in the UTR coverage  [65]. Thus, a large part of the variability observed between the datasets may be attributed to this heterogeneous and low coverage in 3′ regions, even lower in distal isoforms. Very short 3'UTRs In our pipeline, we excluded all 3′ ends occurring within the CDS, but we retained 3′ ends located downstream and very close to the stop codon of Olfr genes (3'UTR length < 100 nt). Some of these very short 3′ ends could actually occur within the CDS given the 100-nt precision window, or could be artifacts due to a dissimilar coverage between the CDS and the downstream sequence.
It should be further noted that the relative abundance of these very short 3'UTRs may be inaccurate, due to the calculation method used. Therefore, the reliability of these very short 3'UTRs (representing 11. 9% of the Olfr genes annotated in both male datasets and 12. 5% for both female datasets) is probably not as strong as that of longer ones. Accordingly, only half of these very short 3'UTRs are conserved in inter-experiment comparisons (51. 3% for males, 49. 3% for females), whereas 75% of the proximal or unique 3'UTRs (without length threshold) are conserved in these comparisons. Fusion transcripts between adjacent Olfr genes Along the development of our pipeline dedicated to Olfr 3'UTRs' characterization, we obtained transcripts corresponding to the fusion of 2 Olfr genes (18-61 Olfr genes concerned depending on the dataset considered; Additional file 2: Figure  S1E-H, ).
No clear evidence for sexual dimorphism in Olfr 3'UTRs We took advantage of the availability of similar male and female datasets to investigate possible sex-specific difference in the repertoires of 3'UTRs of Olfr mRNAs expressed in the adult olfactory mucosa. We observed no sex-specific introns and no global differences in APA, in terms of APA distribution, length of single proximal and distal 3'UTRs, or on the quantitative 3'UTR isoform profiles of Olfr mRNAs between male and female. Moreover, 3 out of the 4 sex-specific pUTRs identified belong to the very short 3'UTR category (< 100 nt for at least one of the datasets), raising the question of their reliability. Therefore, while we cannot rule out the possibility of sex-specific 3'UTR isoforms for some individual Olfr genes, if these isoforms exist, they would involve a small number of these genes.
It has already been shown that the Olfr expression level was highly similar between males and females  [26] , except for the Olfr1347 receptor. This particular Olfr is not annotated in 3 out of the 4 datasets, and the analysis of the coverage argues in favor of a conserved sUTR for Olfr1347 between males and females. Thus, it seems very unlikely that sex-specific 3'UTR isoforms of Olfr mRNAs play a role in sexually dimorphic behaviors linked to odor detection; these behaviors were rather proposed to be due to sex-specific handling of olfactory information by central circuits  [78].
Conclusions The family of odorant receptor genes is the largest gene family in mammals, which provides a unique opportunity to study the diversity and variations of 3'UTRs for a large number of phylogenetically closely related genes within a given species. To study the 3'UTR isoforms of the Olfr genes, we developed a new dedicated pipeline and applied it to RNA-Seq datasets obtained from adult mouse olfactory mucosa of both sexes. The annotations obtained were further validated by multiple experimental approaches on pilot genes, and canonical or variant polyA signals were found associated with these 3′ ends identified in silico. Whereas most of the Olfr genes show variations in 3'UTR length, we detected only rare splicing events downstream stop codons. Thus, we demonstrated that a large number of Olfr genes produce multiple 3'UTR isoforms, mostly generated through alternative polyadenylation.
Strikingly, the diversity in 3'UTRs for the Olfr gene family seems similar to the diversity observed for the whole genome. This alternative polyadenylation is dependent neither on the sex of the animals, nor on the Class I vs. Class II Olfr gene subfamilies. Thus, the determinants for the APA regulation of Olfr genes remain to be investigated. In this study, we chose to explore publicly available RNA-Seq datasets from adult mouse whole olfactory mucosa. While RNA-Seq became the reference method for high-throughput characterization of the transcriptome, alternative dedicated methods now exist to characterized more specifically the 3′ ends of mRNAs, such as 3′-seq  [81]  or PAPERCLIP, even at a cell-type specific level  [82, 83]. However, the pipeline used in the present study could be extended to different situations for which RNA-Seq data is either available or easy to obtain, such as: 1) subpopulation of cells in the olfactory mucosa (e.
Hardware and software This pipeline was constructed on a GNU/Linux operating system (Ubuntu 14) eight cores and 96 GB of RAM, as well as 2 TB of hard drive space. The computational pipeline is a complete bash shell and perl scripts using internal implementation or external software for analyzing RNA-Sequencing, available at https://github. com/doulazmi/Olfr_RNAseq. The analysis steps are expressed in terms of parsing input files to output files as part of the overall workflow (Fig. 2 ).
RNA-Seq data processing and alignment Paired-end read files were aligned to version mm10 of mouse reference genome and transcriptome using STAR 2. 5. 1b [85]  with the following flags -runThreadN 7 --outSAMtype BAM SortedByCoordinate --outSAM-strandField intronMotif --outFilterIntronMotifs Remove-Noncanonical --alignIntronMax 23,000. Threshold for maximal intron size was set to 23 kb, since the largest coherent 5′ intron described for an Olfr in GRCm38. p5 reaches 22. 628 pb (Olfr872). STAR output files in BAM format were sorted and indexed with SAMtools 1. 5  [86]. Preparing a mask file in bed format restricted to the Olfr loci in mouse In order to reduce computational time, and to limit the impact of non-Olfr genes on Olfr transcript assembly, we applied a mask restricted to Olfr genomic regions before IsoSCM and Cufflinks assemblies.