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Fig. 3 | BMC Genomics

Fig. 3

From: Next-generation sequencing-based detection of germline L1-mediated transductions

Fig. 3

Experimental verification of TIGER-based L1-mediated 3′ transductions by PCR. a General primer design: outer (grey arrows) primers were placed outside of the event in the target locus to amplify the L1-mediated sequence transduction insertion allele and/or the reference genome allele. On the left side of the locus, the corresponding sequence (dotted line) uniquely matches the target site, and subsequently matches to multiple positions in the genome in line with the presence of an L1 element. Further to the right, the sequence will also match uniquely to the target site and end with a polyA stretch not seen in the reference genome. In order to confirm the presence and origin of the transduced sequence (source locus), we employed a 2nd set of primers (purple arrows) inside the predicted unique transduction sequence. b Example PCRs verifying rhesus macaque L1-mediated sequence transductions, based on outer primers, are shown for inferred carrier (C) and non-carrier (NC) samples. In the presence of an L1-mediated transduction sequence insertion, a larger band than the reference band in NC is seen; heterozygotes show both bands whereas homozygous L1-mediated sequence transduction insertions show only one (i.e. the higher) band. c A Circos plot shows the distribution for all inferred rhesus macaque L1-mediated sequence transductions (for orangutan and chimpanzee, see Additional file 1: Figure S6); experimentally validated insertions by PCR and MinION single molecule sequencing are depicted in green. Arrowheads indicate directionality towards the target locus

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