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.

^aThe 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/.