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Fig. 3 | Mobile DNA

Fig. 3

From: Genomic approaches to trace the history of human brain evolution with an emerging opportunity for transposon profiling of ancient humans

Fig. 3

Technical challenges of TE insertion profiling for ancient samples. (A) Common features of DNA extracted from ancient humans: 1) Ancient human samples have low-level endogenous aDNA due to DNA contamination; 2) Small amounts of highly degraded aDNA are preserved in ancient bones, teeth, and sediments; 3) Cytosine deamination is a hallmark of base damage in aDNA, resulting in C to T substitution in sequencing data. Uracil-DNA glycosylase (UDG) treatment can be applied to aDNA in pre-library preparation to reduce the base error. (B) WGS-based TE insertion profiling using specialized bioinformatic tools. In modern human WGS data, paired-end reads derived from a polymorphic, non-reference TE insertion are aligned to the reference genome. Tools mainly detect two types of reads near the insertion breakpoints: i) discordant reads (light pink box) that are uniquely aligned to the flanking regions and have their mate-pair reads aligned to many reference TE copies remotely located from the breakpoints, and ii) clipped reads or split reads (light green box) that span the insertion breakpoints and thus have soft-clipped or split mapping to the reference. A read-depth increase due to target site duplication (TSD) is shown. Grey dashed lines indicate the boundaries of TSDs. (C) Characteristics of ancient human WGS data pose challenges in TE insertion detection: limited sequencing depth, short read length, single-end mapping, prevalent C to T substitutions enriched in DNA fragment ends, and occasional contamination of modern human DNA. (D) TE insertion profiling using targeted TE-capture sequencing. Common TE-capture sequencing steps include DNA fragmentation, TE-junction enrichment, next-generation sequencing, read alignment, and insertion calling. DNA fragments originating from TE-junctions are captured and enriched by PCR using TE-specific primers or hybridization using TE-specific probes or microarray. (E) TE enrichment with aDNA extracts is challenging due to a limited amount of endogenous human DNA and highly degraded DNA fragments

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