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. Author manuscript; available in PMC: 2021 Oct 18.
Published in final edited form as: Nature. 2020 Aug 19;584(7821):E29–E33. doi: 10.1038/s41586-020-2523-2

Table 1 |.

Summary of targeted and non-targeted APP PCR methods and lines of evidence that support APP gencDNAs and IEJs

Method Targeted APP PCR Support for the existence of IEJs and gencDNAs Reference
Approaches without targeted APP PCR
1 RISH on IEJ 3/16 None IEJ 3/16 RNA signal is present in human SAD brain tissue Lee et al.2
2 Whole-transcriptome SMRT-seq None An independent commercial source identified IEJs in APP and other Public dataseta, genes Lee et al.2this Reply
3 Targeted RNA SMRT-seq None RNA pull-down that identified APP IEJs Public dataseta, Lee et al.2
4 DISH of gencDNAs None IEJ 3/16 and exon–exon junction 16/17 showed increases in neurons compared to non-neurons from the same brain from an individual with SAD and to non-diseased neurons; J20 mice containing the APP transgene under a PDGF-β-promoter showed increased number and size of signal compared to non-neurons and wild-type mice Lee et al.2
5 Dual point-paint FISH None Identified APP CNVs of variable puncta size that were not always associated with Chr21 Bushman et al.3
6 PNA-FISH None APP exon copy number increases show variable signal size and shape with semiquantitative exonic probes Bushman et al.3
7 Agilent SureSelect targeted pull-down None Identified APP gencDNAs in brains from individuals with SAD; contains plasmid sequence contamination Lee et al.2,this Reply
New #7 Agilent all-exon pull-down None All-exon pull-downs, with no plasmid contamination by both Vecscreen and Vecuum, contain APP gencDNA sequences and evidence of gencDNA UTRs and novel insertion sites Park et al.4, this
Reply
Approaches with targeted APP PCR
8 RT–PCR and Sanger sequencing Oligo-dT primed and targeted APP primers Novel APP RNA variants with IEJs; predominantly in neurons from individuals with SAD Lee et al.2
9 Genomic DNA PCR and Sanger sequencing Yes Identified APP gencDNAs with IEJs; predominantly in neurons from individuals with SAD Lee et al.2
10 Genomic DNA PCR and SMRT-seq Yes IEJ/gencDNAs were more prevalent in number and form in neurons from individuals with SAD compared to non-diseased neurons; identified 11 pathogenic SNVs that were present only in SAD samples Lee et al.2
11 APP-751 overexpression in CHO cells Yes IEJ and gencDNA formation required DNA strand breakage and reverse transcriptase Lee et al.2
12 Single-cell qPCR Yes; individual exon Intragenic exon 14 single-cell qPCR showed copy number increases in prefrontal cortical neurons over cerebellar neurons from the same brain of an individual with SAD Bushman et al.3

CNV, copy number variation.

a

The Alzheimer brain Iso-Seq dataset was generated by Pacific Biosciences, Menlo Park, California. Additional sequencing information and analysis is provided at https://downloads.pacbcloud.com/public/dataset/Alzheimer_IsoSeq_2016/.