Nature - USA (2020-08-20)

Nature | Vol 584 | 20 August 2020 | E21

Non-PCR-based hybrid-capture sequencing

Read mapping

...

... Clipped backbone sequenceAPP vector

Reference

genome

...

...

...

...

PCR-based assays: cloning, SMRT-seq

Source APP

(sample DNA)

APP insert

(vector contaminant)

No PCR product

... owing to large gene size

...

APP(sample DNA) retrocopy ...

5 ′ UTR 3 ′ UTR

AAAA

Indistinguishable

PCR primers

targeting coding exons

NeuN+ neuronal nuclei

APPcoding exons only insert with

...

......

...

APP coding sequence end APP coding sequence start

Clipped vector

backbone sequence

Reference

genome

(GRCh38)

APP vector contamination

Source APP

APP insert

(vector contaminant)

APP retrocopy ... AAA

Source APP site

PCR

exon

intron

...

...

...

...

...

...

Paired-end

sequence reads

... AAA Clipped retrocopy

target site sequence

5 ′ UTR 3 ′ UTR

Vector (pGEM-T Easy)

backbone sequence

CGCGAATTCACTAGTGAT

GCGCTTAAGTGATCACT A

3 ′ T overhang

T

A APP insert

AATCGAATTCCCGCGGCCG

TTAGCTTAAGGGCGCCGGC

3 ′ T overhang

APP insert

a

b

3 ′UTR 5 ′UTR

AAA

AGGCTGCTGTGGCGGGGGTCTAGTTCTGCATCTGCTC

********** NNNNNNNNNN QQQQQQQQQQ MMMMMMMMMM QQQQQQQQQQ EEEEEEEEEE

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGATT

CCGGCCGGAAAATTTTCCAACCTTAAGGTTGGAATT

C G T

TGCCAAACCGGGCAGCATCGCGACCCTGCGCGGGGCA

G AAAATTCCGGAAAATTTTCCCCCCGGCCGGGGCCCCGG

AAAATTCCGGAAAATTTTCCCCCCGGCCGGGGCCCCGG

AAAATTCCGGAAAATTTTCCCCCCGGCCGGGGCCCCGG

AAAATTCCGGAAACTTTTCCCCCCTGCCGGGTCCCCGG

AAAATTCCGGAAAATTTTCCCCCCGGCCGGGGCCCCGG

AAAATTCCGGAAAATTTTCCCCCCCCGCGGGCCCCCGG

AAAATTCCGGAAAATTTTCCCCCCGGCCGGGGCCCCGG

CC AATCGAATTCCCGCGGCCG

AAAAAAAA LLLLLLLL GGGGGGGG PPPPPPPP LMLMLMLMLMLMLML M

2,000

1,000

650

400

200

1–181–18N2–17

APP-751 APP-695

Restriction sites BstZINotIEcoRI SpeI EcoRI SacII BstZINotI

c

25,881,660 bp 25,881,670 bp 25,881,680 bp 26,170,610 bp 26,170,620 bp 26,170,630 bp

0

5

Clipped r

ead fraction (%)

123456791011 1213 1415161718

Exons

10

15

20

Estimated by APP vector clipped sequences

Expected fraction estimated frExpected fraction estimated from the Lee study DISH experiment om the Lee study DISH experiment

1–181–18N2–17 Sequence homology between two junctions

R6/17

CA

CA

Exon2 CA

...CC Exon17

AT...

R2/17

R6/18

R3/14

R3/17

AGCCAAC

AGCCAAC

AGCCAAC

...GA

AC...

Exon14

Exon3

GCAGTG

GCAGTG

GCGGTG

...AA

TT...

Exon17

Exon3

GAGGA

GAGGA

GAGGA

...AC

GC...

Exon18

Exon6

AGATGGGAGTGAAGACAAAG

AGATGGGAGTGAAGACAAAG

AGATGTGGGTTCAAACAAAG

...GC

GT...

Exon17

Exon6

R2/16 AT

AT

AT

...GT

GC...

Exon16

Exon2

R2/14 ACCAAGGA

ACCAAGGA

ACCAAGGA

...AT

TC...

Exon14

Exon2

R1/14 GCTC

GCTC

GCTC

...CG

CT...

Exon14

Exon1

Fig. 1 | APP vector contamination in the Lee study. a, APP vector contamination
and its manifestation in genome sequences. PCR-based assays in the Lee study^2
fail to distinguish between APP retrocopy and vector APP insert. Hybrid-capture
sequences from the Lee study show clipped reads with a vector backbone
sequence (pGEM-T Easy), including restriction sites at the multiple cloning site
and a 3′ T-overhang. b, Estimated fractions of cells with APP gencDNA at the exon
junctions in the Lee hybrid-capture data. All exon junction fractions (black dots)
are comparable to the fraction at the coding sequence ends with vector

backbone sequences (red dots). The dotted line above represents the

conservative estimate of expected fraction based on the Lee DISH experiment

(see Supplementary Methods); shaded area, 95% confidence interval.

c, Electrophoresis and sequencing of PCR products from the vector APP inserts

(APP-751/695) showing new APP variants as artefacts. Eight out of twelve IEJs

found both in our APP vector PCR sequencing and the Lee study RT–PCR results

are shown (Extended Data Fig. 3).