Novel MEI events. Novel MEI events, as detected by WGS on the CG platform, in 100 samples (detailed in ). (A) Total novel L1Hs. High numbers were detected in the brain samples and particularly in the SEGA, Rett, AT and NSA samples. The y axis shows the number of novel L1Hs insertion events. (B) Total novel AluY. (C, D) The polymorphic novel L1Hs (C) and AluY (D) insertions we identified by CG sequencing, that were also found in the 1000 Genomes Project data. Note the uniformity of numbers of novel polymorphic germline insertions in the various samples. The normal LB samples with the highest number of polymorphic L1Hs and AluY belong to the YRI population and are highlighted in blue. (E, F) Somatic insertions of L1Hs (E) and AluY (F) are shown for all the samples we analyzed. Somatic L1Hs and AluY were high mainly in the brain samples particularly in AT, NSA, Rett and SEGA samples. Note the scale break in the y axis in (A) and (E) and the different scales in the different panels. The x axis is common to all the panels: AT, ataxia telangiectasia; NSA, non syndromic autism; Rett, Rett syndrome; SEGA, TSC-specific subependymal giant cell astrocytomas; PB, Peripheral Blood; TSC, TSC non tumoral brain sample; NB, normal brain; LB, normal lymphoblastoid cell lines representing various ethnic populations. Population name abbreviations: ASW, African ancestry in Southwest USA; CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; CHB, Han Chinese in Beijing, China; GIH, Gujarati Indian in Houston, Texas, USA; JPT, Japanese in Tokyo, Japan; LWK, Luhya in Webuye, Kenya; MKK, Maasai in Kinyawa, Kenya; MXL, Mexican ancestry in Los Angeles, California; TSI, Toscans in Italy; YRI, Yoruba in Ibadan, Nigeria; PUR, Puerto Rican in Puerto Rico; P (PDRP), an individual from the polymorphism discovery resource; T: (TRIO), two parents and one child.

Structure of L1Hs insertion events as detected by CG analysis and verified by PacBio single-molecule sequencing. (A) Illustration of nested (right) and non-nested (left) L1Hs insertions. Nested insertions are those integrating into a pre-existing genomic repetitive element. The retrotransposon (red) can be inserted into a unique genomic region or land in a preexisting repetitive element (green). (B, C) depict a non-nested insertion event; (D, E) depict a nested insertion event. (B) an example of a non-nested insertion in chromosome 1. Chromosomal location of a novel L1Hs insertion identified in the SEGA1 sample is shown as a vertical red line and the genomic coordinates are shown according to the UCSC chromosome chart. Detection of the non-nested MEI was by CG paired-end reads (DNBs): red arms map to the reference genome (hg19) and blue arms map to the L1Hs canonical sequence. The arms are separated by ∼400 bp spacers (black line). The region highlighted in yellow shows the L1Hs insert that is not present in the hg19 reference genome (See details in ). Forty out of the 270 paired end reads (DNBs) supporting this insertion event are shown here. The full coverage is given in . (C) The detailed mapping by the PacBio platform of the same 203 bp long L1Hs insertion that is depicted in (B). The inserted retrotransposon includes the 5 832 to 6 035 L1Hs fragment, a poly A tail and is flanked by target-site duplication (TSD) sequences (light blue boxes). (D) Chromosomal location and genomic coordinates of a novel L1Hs insertion nested into a preexisting LIPA4 element in chromosome 11. Fifty-seven out of the 287 supporting reads are shown. The full coverage is depicted in . The sequence of this L1PA4 repeat in the reference genome diverged enough to represent a unique sequence, and reads could map to the insertion in this repetitive element location with high score (). (E) The detailed mapping by PacBio of the 347 bp long L1Hs insertion nested in the L1PA4 repeat element depicted in (D), includes the 5 701 to 6 048 canonical L1Hs fragment, a poly A tail and is flanked by TSDs (light blue boxes). In this case the left TSD contains a T nucleotide not present in the reference sequence (marked in grey). (F) Number of somatic L1Hs insertions in the analyzed samples. The y axis shows the number of L1Hs. The non-nested insertions in each sample are marked in yellow.

Structural characterization of inserted L1Hs insertions and enriched motifs at L1Hs and AluY insertion sites. (A) Schematic illustration of the main types of novel L1Hs insertions: The coordinates of the canonical 6 070 bp long L1Hs sequence (broken line) are presented. The bars depict examples of intact and truncated inserted elements. Insertion types were defined based on stringent criteria: ≥ 10 supporting reads (DNBs) for each end, shortening of > 500 bp from the canonical 5′ end and of > 570 bp from the canonical L1Hs 3′ end (broken vertical lines). (B) The percentage of L1Hs insertions truncated at the 5′ end, in the brain samples. (C) The percentage of L1Hs insertions truncated at the 3′ end, in the brain samples. (D) The percentage of L1Hs insertions truncated at both 5′ and 3′ ends, in the brain samples. (E) The most enriched motifs detected at the insertion sites of somatic and germline L1Hs in the brain and in the normal LB samples. In all groups, except the brain nested L1Hs insertions (where a novel motif was identified), the most enriched motif is the known L1 EN cleavage sequence (YY/RRRR).

Genes with somatic L1Hs insertions. (A, B) Enriched functional groups of genes with L1Hs insertions. Genes that were inserted with at least one somatic L1Hs event were analyzed for functional group enrichment. Enriched neuronal function gene groups (A) and neurological and psychiatric disorder gene groups (B) with more than 20 genes and P ≤ 5.0 ×10⁻³ (Ingenuity IPA; Fisher's exact test; Benjamini-Hochberg) in the brain samples analyzed. (C, D) Box plot charts of the number of nested (C) and non-nested (D) somatic L1Hs insertions in genes as distributed in each of the various neurodevelopmental disorders and the normal brains: AT (n = 4), NSA (n = 2), Rett (n = 2), SEGA (n = 3), TSC (n = 4) and NB (n = 5). Non nested L1Hs insertions are more abundant in the AT, NSA and Rett pathological samples. (E, F) Examples of genes associated with neurological and psychiatric disorders with nested (E) and non nested (F) L1Hs somatic insertions. The L1Hs insertions examples (orange bars) are located in introns (exons are marked as dark blue bars) and are nested (E) in pre-existing L1 family members (blue box) in the same orientation as the host element (orange and blue arrows for the novel L1Hs and the host L1, respectively), and opposite to the gene orientation (black arrows) in most of the cases. The non-nested L1Hs insertions (F), are also inserted opposite to the gene orientation. The non-nested insertions in FAM126A and OPHN1 are examples of exonic insertions.