dataesr/scientific-paragraphs-categorization huggingface dataset:
, 2002; Smit, 1999). Although, the median expression level of genes with new L1 insertions is higher than for the Random and BMC datasets, it is not significantly different from that of genes containing L1 target site motifs (MMC, Figure  5B ). Moreover, the slight association between higher gene expression levels and L1 insertions is much less pronounced than observed for other transposable elements or retroviruses, in particular HIV, known to preferentially integrate into highly expressed genes  (Sultana et al. , 2017). L1 retrotransposons inserted within genes and recorded in the human reference genome occur more frequently in the opposite orientation with respect to their enclosing gene  (Medstrand et al. , 2002; Smit, 1999; Zhang et al. , 2011). Since a majority of disease-causing L1 insertions are in the sense orientation relative to the disrupted gene  (Hancks and Kazazian, 2016) , a generally accepted explanation is that sense insertions are more likely to be detrimental and counter-selected after integration.
, 2017; Hansen et al. , 2010; Hiratani et al. , 2008). This enrichment is particularly evident for more recent, primate-and human-specific L1 subfamilies (Figure  S7A ). To directly investigate whether this reflects preferential L1 insertion into late-replicating regions, we computed the association of new L1 insertions with replication timing, and with other genomic properties linked to DNA replication such as origins of replication and replication fork directionality (RFD), using publicly available datasets.
L1 integration is influenced by host DNA replication We observe that new L1 insertions occur preferentially within early-replicating regions of the genome (Figure  6A , left, and 6B). This represents the strongest association with any genomic feature analyzed in this study, and is sufficient to explain the detectable skew of L1 insertions in other largescale domains (including lamin-associated domains (LADs) and topological A and B domains; see Figure  S5A ). Integration sites recovered without selecting for the reporter cassette are also enriched in early replicating regions and their replication timing markedly differs from that of endogenous L1 elements, although this is less pronounced than for selected events (Figure  S7A ). Neither local basecomposition, nor the density of L1 target motifs at the insertion sites (BMC and MMC control datasets, Figure  6B ), nor the increased copy number of already-replicated regions (Figure  S7B ), are responsible for this association.
DISCUSSION Finally, our strategy of analyzing de novo L1 integration patterns in a single, well-studied cell line was designed to benefit from the availability of a broad range of genomic and epigenomic datasets. Although these data generally come from the same cell line, they derive from independent cell stocks cultured in conditions that might differ from those used in our L1 retrotransposition assay. Despite this limitation, we could corroborate our findings using multiple external datasets (Figures  S2, S5C ), and we validated the most-significant detected association with a histone mark (H3K4me1; Figures  4C, S5C ) using data generated from our own cell stocks and culture conditions (Figure  S5D-E ). However, it is conceivable that some aspects of L1 transposition may also be influenced by cell linespecific factors, which our analysis could overlook.
We conclude that mapping de novo insertions, rather than existing insertions that accumulated in the genome over a long period of time or were selected by clonal expansion during tumor growth, is essential to separate insertion site preference from evolutionary selection. In sum, our data reveal the unique properties of the L1 machinery which allow this transposable element to broadly target most regions of the human genome. They also highlight how the landscape of insertions is driven by pre-existing aspects of genomic sequence and how it is reshaped throughout evolution by selection. In a more general way, our work illustrates the co-adaptive trajectories of transposable elements with their host. (A) Experimental workflow. De novo L1 retrotransposition was induced by transfecting HeLa S3 cells with a plasmid-borne L1 element containing a neomycin-based retrotransposition marker (see Methods).
(D) Distribution of motif scores based on the position weight matrix (PWM) shown in (C) for observed (L1 neo, blue) or random (red) datasets. Motif scores are binned by 0. 5 and the curves represent Gaussian fits. The L1 target motif (as defined in panel C) is present at the vast majority of the mapped insertion sites in contrast to sequences found at random sites (p<1e-16, Wilcoxon test). See also Figure  S1. Heat maps and average profiles of nucleosome occupancy around pre-integration (L1 neo) or control sites (MMC, BMC and Random) as measured by MNase-seq in HeLa S3 cells [data from  (Hansen et al. , 2010) ]. For each heat map independently, loci were sorted by increasing signal in the central 150 bp-window. Average nucleosome densities were calculated either globally (bottom graphs, blue) or by separating them in three classes of equal size, after ranking them by increasing levels of nucleosome density at the insertion or control sites (green graphs; low, middle and high nucleosome occupancy, from top to bottom).
The high nucleosome occupancy class indicates that nucleosome presence per se does not prevent L1 integration. Global profiles of controls on the bottom represent the average of 50 datasets. MMC, motif-matched control; BMC, basecomposition-matched control. See also Figure  S2. (A) L1 integration sites are dispersed in all chromosomes. Each dot represents a chromosome. The number of L1 integration sites is highly correlated (Pearson correlation) with HeLa S3 whole genome sequencing (WGS) coverage of each chromosomes (number of reads). Chromosome 1 is more frequently targeted than expected (red dot, two-tailed binomial test, FDR-adjusted p=0. 010). (B) Spacing between de novo L1 insertions. Empirical cumulative distribution of target site spacing (bp) was compared with those of in silico generated random datasets.
L1 neo (black), count of observed L1 insertion sites. The overlaid blue line represents the average number of potential L1 target sites (average count of motif-matched control sites per bin, 1565 MMC sampled 1000 times). Red bars indicate hotspots (Poisson distribution corrected for CNV, FDR-adjusted p < 0. 05). See also Figure  S3  and Table  S3. (A) Association of various transposable elements and retroviruses with ENCODE chromatin states. The 18 chromatin states were defined by ENCODE based on a combination of several histone marks (ChromHMM). Heat map displays the magnitudes of overlaps with each chromatin state, expressed as z-scores (see methods, blue for depletion and red for enrichment). Publicly available de novo insertion datasets previously obtained for other classes of elements were analyzed in parallel  (Gogol-Döring et al.
, 2016; LaFave et al. , 2014; Wang et al. , 2007). Endogenous L1s (L1 endo) correspond to the 3' ends of endogenous L1HS-Ta elements present in the HeLa-S3 genome  (Philippe et al. , 2016). Chromatin segmentation data were obtained from ENCODE in HeLa S3 (for L1 and simulated datasets) or K562 cells (for SB, HIV, PB and MLV). BMC, MMC and Random are in silicosimulated control datasets (see Figure  2 legend ). SB, Sleeping Beauty; PB, PiggyBac; MLV, Murine Leukemia Virus; HIV, Human Immunodeficiency Virus. (B) Proportion of insertions falling in each chromatin segment for de novo insertions compared to endogenous L1HS-Ta copies and to a Random dataset. (C) Association of various transposable elements and retroviruses with histone modifications. Heat map displays z-scores of observed overlaps with various histone ChIP-seq peak obtained by ENCODE in HeLa S3 (for L1 and simulated datasets) or K562 cells (for SB, HIV, PB and MLV).
Color-scale and legends are as in (A). (D) The tempo of primate-specific L1 association with a subset of functional chromatin segments. EnhWk, weak enhancer; TxWeak, weak transcription; Quies, quiescent chromatin segments. L1HS is equivalent to L1PA1. L1 are sorted by increasing age  (Khan et al. , 2006). Curves represent loess-smoothed profiles of z-scores across primate-specific L1 subfamilies. Solid and dashed red lines indicate the z-score values of L1 neo and endogenous L1 elements as a whole, respectively. See also Figure  S4 , Figure  S5 , and Figure  S6. (A) Proportion of various transposable elements and retroviruses inserted in genes. Values and error bars for simulated controls represent the average percentage of 1000 datasets ± s. d. The dashed line at 45% indicates the proportion of the human genome encompassed by annotated protein coding genes (GENCODE v19).
A red bullet indicates a significant difference with this proportion (p<0. 001), while a grey bullet indicates no significant difference (p≥0. 05) in a two-tailed binomial test with FDR correction (FDR<0. 05). (B) Expression levels of genes containing insertions. Each dot represents the median expression level of genes with an insertion in each of the 1000 simulated datasets, overlaid with their median and interquartile range Sultana, van Essen et al. 2019 -p. 26/37 (red). On the right, the median expression levels of genes with experimental insertions are indicated. With the exception of endogenous L1HS-Ta (L1 endo), all tested elements are inserted in genes with significantly higher levels of expression than expected randomly (L1 neo vs Random, p=0. 012; L1 neo vs BMC, p=0. 032; all others p<0.
002). However, the median expression level of genes with novel L1 insertions does not significantly differ from those containing L1 target motifs (L1 neo vs MMC, p=0. 204). p-values are two-tailed estimates from empirical distributions. TPM, transcripts per million. Poly(A) + RNA-seq data in HeLa S3 were obtained from ENCODE. (C) Orientation of L1 insertions relative to genes. Both de novo (L1 neo) and endogenous L1 insertions occur significantly more frequently in antisense direction with respect to gene transcription (two-tailed binomial test). (D) and (E) Biased de novo L1 insertion orientation results from an asymmetric distribution of L1 target motifs in transcribed vs non-transcribed strand. A/S ratio, ratio of insertions in antisense vs sense orientation relative to genes. Each dot represents the mean A/S ratio in each of the 1000 simulated datasets, overlaid with their median and interquartile range (red).
On the right, the A/S ratios for experimental datasets are indicated. Since Random, BMC, SB, PB and HIV datasets were unstranded, orientations were assigned randomly to each insertion and their average ratio is expected to be equal to 1. (E) A/S ratios comparisons between experimental and control datasets. p-values are two-tailed estimates from empirical A/S ratio distributions. See Figure  4A  for dataset definitions. (A) Expected distribution (histogram) and observed (line) average signal at L1 pre-insertion loci for waveletsmoothed repli-seq signal (Replication timing, left), origins of replication (SNS-seq peak height; ORI, middle), and replication fork directionality signal (measured by OK-seq; RFD, right). The expected distributions represent the distribution of the mean signal of 1565 randomly-selected genomic positions, repeated 900 times.
EXPERIMENTAL MODEL AND SUBJECT DETAILS HeLa S3 cells were obtained from ECACC (distributed by Sigma-Aldrich) and maintained in a tissue culture incubator (37°C at a 5% CO2 level) in Dulbecco's modified Eagle medium (DMEM) containing 4. 5 g/L D-Glucose, 110 mg/L Sodium Pyruvate, and supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Growth medium was also supplemented with 862 mg/mL L-Alanyl-L-Glutamine (Glutamax). Cell cultures tested negative for mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza). Cell line authenticity was verified by multiplex STR analysis (PowerPlex 21 PCR system, Promega, assays performed by Eurofins Genomics as a service provider) and comparison with the DSMZ database (https://www. dsmz. de/services/services-human-andanimal-cell-lines/online-str-analysis.
Plasmid constructs The plasmid pOS24 consists in a full length and retrotransposition-competent human L1 (L1. 3 clone, Genbank accession number L19088), containing the mneoI retrotransposition reporter cassette  (Figure 1A and (Moran et al. , 1996) ). The engineered L1 was cloned into a modified pCEP4 backbone (Invitrogen) in which the CMV promoter and SV40 polyadenylation sequence were removed. Hence, L1 is expressed from its natural internal promoter (in the 5'UTR) and transcription is terminated by its natural polyadenylation signal. The mneoI cassette is expressed from an SV40 promoter and allows discriminating de novo copies from the thousands of endogenous L1 elements already present in the human genome. Neomycin (G418)-resistance was used to enrich cells containing new retrotransposition events (L1 neo).
For each sample, 9 parallel reactions were performed. Droplets of one reaction were read with a QX200TM droplet reader and analyzed with the QuantaSoft software to control amplification. Droplets from the remaining 8 reactions were pooled and amplified DNA was extracted from the droplets by the chloroform extraction method according to manufacturer's protocol. Size selection. Pooled amplicons from either PCR method were subjected to double size selection to retain amplicons ranging between 300 and 450 bp by two consecutive Agencourt AMPure XP bead purification steps using beads-to-DNA ratios of 0. 6:1 and 0. 7:1, respectively. The supernatant of the first bead purification using bead:DNA ratio of 0. 6:1 contains DNA fragments larger than 300 bp. This supernatant is applied to a second selection step with a bead:DNA ratio of 0.
ACKNOWLEDGMENTS We are grateful to  C. Baudoin  and  IRCAN Genomics Core Facility  for sequencing (supported by  FEDER ,  Région Provence Alpes Côte d'Azur ,  Conseil Départemental 06 ,  ITMO Cancer Aviesan  (plan cancer) and Inserm), and to  O. Croce  and IRCAN bioinformatics service for computing resources. We are grateful to the  ENCODE Consortium  for RNA-seq, ChIP-seq and Repli-seq data. This work was supported by a joint grant to SS and GC from the  Fondation pour la Recherche Medicale  ( FRM-DEP20131128533 ), by grants to GC from the  European Research Council  ( ERC-2010-StG 243312 , Retrogenomics), the  Agence Nationale de la Recherche  ( LABEX SIGNALIFE ,  ANR-11-LABX-0028-01 ;  RETROMET ,  ANR-16-CE12-0020 ), the  Canceŕopole PACA (Projet Emergence) ,  CNRS  ( GDR 3546 ), and the  University Hospital Federation (FHU) OncoAge.
TS was supported by a joint  Erasmus Mundus Mobility  with  Asia (EMMA)  fellowship between  Université Cote d'Azur (France)  and  University of Sultana , van Essen et al. 2019 -p. 17/37 Dhaka (Bangladesh). Work in the laboratory of NG was supported by the  Agence Nationale de la Recherche  ( ANR-12-BSV6-0003 , RETROGENO).
Identification of alternative splicing events To characterise alternative splicing at the PALB2 locus, we analysed RNAs isolated from 112 specimens, including lymphoblastic cell lines not treated with the NMD-inhibitor puromycin (n=68), matched replicates treated with puromycin (LCLs+Puro, n=1), stimulated leucocytes cultures not treated with puromycin (n=6), matched replicates treated with puromycin (sLEU+Puro, n=3), RNA stabilised peripheral blood samples (PAXgene, QIAGEN, n=7; Tempus, Thermo Fisher, n=10), non-malignant breast tissue samples from unrelated women (Breast, n=12; 10 corresponding to women with a diagnosis of breast cancer, of which 9 are included in SCAN-B, ClinicalTrials. gov identifier: NCT02306096; 2 corresponding to women without a diagnosis of breast cancer included in CASOHAR trial NTC02560818), a human mammary epithelial cell (HMEC, n=1, 2 technical replicas included in the analysis), commercially available RNA from non-malignant breast tissue (Clontech 636576, n=1), normal ovarian fimbriae tissue samples from prophylactic oophorectomies performed in postmenopausal women without cancer (Fimbriae, n=2) and one pool of 3 non-malignant ovarian tissues (Clontech 636555, n=1).
Annotation of alternative splicing events We described all alternative splicing events according to HGVS guidelines, using as a reference the Ensembl transcript ENST00000261584. 8 (NCBI RefSeq NM_024675. 3). For the sake of simplicity, we also identified most events with a code that combines the following symbols: ∆ (skipping of reference exonic sequences), ▼ (inclusion of reference intronic sequences), E (exon), I (intron), p (acceptor shift), q (donor shift), AFE (alternative first exon) and IVS± (located at intervening sequence). When necessary, the exact number of nucleotides skipped (or retained) is indicated. Events were annotated as well according to the confidence of the finding (high-confidence vs lower-confidence), predictions on coding potential (LoF vs uncertain) and relative quantification (expression level relative to the corresponding reference transcript) (see online supplementary material section 2 and figures 2-5 for further details).
the same percentage was observed in whole transcriptome rna experiments, although the total number of reads was much lower (14 933 reads combining data from laboratories 2 and 3). rt-Pcr/ce contributed 1747 data points (individual rt-Pcr experiments performed with a particular combination of primers in individual samples, including technical replicas). cloneSeq analysis contributed 1. 2×10 6 reads (≈2. 4% of the reads supporting alternative splicing events). Data were pooled together, reviewed and cross-checked to end up with a list of high-confidence naturally occurring alternative splicing events (events detected by different techniques in different samples), and a list of lower-confidence splicing events (events not qualifying for higher confidence events). Finally, the possible relevance of high-confidence findings for the initial classification of canonical splicing site and Ptc-nMD variants was explored.
acMg-aMP, american college of Medical genetics and genomics-association for Molecular Pathology; HMec, human mammary epithelial cell; lcl, lymphoblastic cell line; nMD, nonsense-mediated decay. analysis data that was independently produced at five contributing centres (figure  1 ). The analysis identified 44 naturally occurring alternative splicing events with high-confidence (online supplementary table 1) and provided evidence for the existence of up to 44 additional (lower-confidence events, online supplementary table 2 and supplemental material section 2. 2). Most events (37 out of 44 high-confidence and all lower-confidence events) have not been described previously in GENCODE (https://www. gencodegenes. org/) or the scientific literature to our knowledge. Up to 15 high-confidence events preserved a bona fide open reading frame (ie, an ORF spanning from the reference start codon to the reference termination codon, table 1, protein column).
hgvs. org/, using Ensembl transcript ENST00000261584. 8 as a reference. §Uncertain coding potential if the transcript encodes a protein predicted to preserve (or partially preserve) functional capacity. See online supplemental material section 2. 3 and figure  4  for further details. ¶Only▼(AFE600)+∆(E1) described in GENCODE (comprehensive gene annotation from GENCODE release 26 retrieved through Ensembl at http://www. ensembl. org/). **Δ(E6) transcripts code for a hypomorphic protein (instable, but with residual activity). 26 †Only Δ11_12 described previously in the literature. 12 C, N-terminal coiled-coil domain; ChAM, chromatin-associated motif; LOF, loss-of-function; WD40, WD40 β-propeller C-terminal domain.
Alternative splicing analysis might be influenced by many factors, including collection of RNA samples, experimental design and detection sensitivity. For instance, one study characterising alternative splicing at breast cancer susceptibility genes by RNAseq noticed the poor performance of PAXgene if compared with LCL samples,  12  and a previous ENIGMA collaborative study comparing RT-PCR splicing protocols across different laboratories concluded that primers design and detection sensitivity (rather than RNA extraction and/or cDNA synthesis protocols) had an impact on the †Biotype according to ENCODE. 25 NA described according to the Human Genome Variation Society rules at http://varnomen. hgvs. org/, using Ensembl transcript ENST00000261584. 8 as a reference. §described previously in the literature.
kconFaB provided research resources used in this study. aBS coordinated the enigMa consortium. aB, eaV, MPV, PD, aK, aBS, lW and SK contributed to the conception and design of the study, contributed to obtain all necessary approvals and clearances to conduct the research, contributed to data acquisition, contributed to data analysis, contributed to grant funding, revised the manuscript for important intellectual content and approved the final version. MdH contributed to obtain all necessary approvals and clearances to conduct the research, contributed to the conception and design of the study, contributed to data acquisition, contributed to data analysis, contributed to grant funding, wrote the manuscript and approved the final version. Funding PD, MPgW, eaV, aB, aK and MH have received funding from the european Union's Horizon 2020 research and innovation programme under grant agreement no.
634935. rl is supported by a normandy-University, Federation-Hospitalo-Universitaire (FHU) grant. Vl is supported by a Mackenzie Family cancer Postdoctoral Fellowship. rB is supported by funding from the european Union's Horizon 2020 research and innovation programme under grant agreement no. 634935. aM is supported by a French cancéropôle nord-Ouest (cnO) grant. aB is supported by Mrs Berta Kamprad Foundation. eaV and MH are supported by Spanish instituto de Salud carlos iii (iSciii) funding (grants Pi17/00227 to eaV and Pi15/00059 to MH), an initiative of the Spanish Ministry of economy and innovation partially supported by european regional Development FeDer Funds. aBS is supported by an nHMrc Senior research Fellowship (iD1061779). lcW is supported by the rutherford Discovery Fellowship.
4-IgH-Trastuzumumab using Gibson Assembly cloning (NEB)  66  and were produced in Expi293 cells (Invitrogen) grown for 5 days at 37 °C with 8% CO 2. All IgG variants proteins purified by Protein A high capacity agarose resin (Thermo Scientific) according to the manufacturer's instructions. Purity of antibody variants was confirmed by SDS-PAGE and size exclusion chromatography and was over 95%. Library construction and screening. Amino acid residues D249-R255, V308-L314, and E430-Q438 in the Fc that are within 7 Å distance from FcRn in the crystal structure of the human Fc:FcRn complex (PDB ID: 4N0U)  13  were selected for mutagenesis using the aa substitution scheme shown and three separate libraries were constructed using primers with degenerate codons (Supplementary Table  1 ). The heavy chain of trastuzumab IgG1 was used as the template.
A fast gradient of 0. 1% formic acid/water and 0. 1% formic acid/acetonitrile over 10 min was used to elute the intact proteins from an OPTI-TRAP TM protein microtrap (Optimize Technologies). The Orbitrap Fusion was operated in Intact Protein Mode at 15,000 resolution from 400 to 2000m/z. The data were deconvoluted using Thermo Protein Deconvolution software. Size exclusion chromatography. SEC analyses for the purified IgG proteins were performed on an ÄKTA Pure (GE Healthcare) liquid chromatography system using a Superdex 200 10/300GC, (GE Healthcare), with a mobile phase of pH 7. 4 PBS at a flow rate of 0. 75 mL/min. Chromatograms were obtained by monitoring the absorbance at 280 nm. The injection amount was 100 μg of proteins in a volume of 200 μL. For thermal stress test, each antibody protein (1 mg/mL) was incubated at 70 °C for 10, 20, 40, and 60 min and then analyzed by SEC using the above described method.