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Targeted sequencing with the Ion Torrent System is able to identify single nucleotide variants, small insertions and small deletions. Variants in repeat sequences, large homopolymers and large insertions/deletions are not or difficult to identify.
The RBC Enzym AmpliSeq Panel v1 (IAD95230_241) consists of 327 amplicons and is covering 99.48% of desired regions (all coding regions (exons), flanking intronic regions, untranslated regions and promotor areas with known (likely) pathogenic variants) from the 22 genes listed in Table 4.

Table 1: List of submitted genes and the coverage of the designed panel

Gene
Chromosome
NCBI Transcript
Exons
Coverage
Coverage 5' of initiation site (flanking sequence and 5'UTR)
Amplicons
ABCB7
chrX
NM_004299.4
16
100,00%
-22 relative to initiation codon
24
AK1
chr9
NM_000476.2
6
100,00%
-87 relative to initiation codon
9
ALAS2
chrX
NM_000032.4
11
100,00%
-240 relative to transcription initiation site
19
ALDOA
chr16
NM_001243177.1
10
100,00%
-8 relative to initiation codon
12
CYB5R3
chr22
NM_001171660.1
9
97.25%
-189 relative to initiation codon
12
ENO1
chr1
NM_001428.
12
100,00%
-119 relative to initiation codon
12
G6PD
chrX
NM_000402.4
13
100,00%
-176 relative to initiation codon
19
GAPDH
chr12
NM_002046.5
9
77.48%
-36 relative to initiation codon
8
GCLC
chr6
NM_001498.3
16
100,00%
-713 relative to initiation codon
22
GPI
chr19
NM_000175.3
18
100,00%
-79 relative to initiation codon
22
GSR
chr8
NM_000637.3
13
98.94%
-115 relative to initiation codon
15
GSS
chr20
NM_000178.2
13
100,00%
-151 relative to initiation codon
15
HK1
chr10
NM_033497.2
22
100,00%
-157 relative to initiation codon
27
NT5C3A
chr7
NM_001002010.2
9
100,00%
-60 relative to initiation codon
13
PFKM
chr12
NM_001166686.1
25
100,00%
-129 relative to initiation codon
22
PGD
chr1
NM_002631.2
13
98.93%
-175 relative to initiation codon
13
PGK1
chrX
NM_000291.3
11
100,00%
-75 relative to initiation codon
12
PKLR
chr1
NM_000298.5
11
100,00%
-305 relative to initiation codon
16
PRDX2
chr19
NM_005809.5
6
100,00%
-48 relative to initiation codon
5
UGT1A1
chr2
NM_000463.2
5
100,00%
-124 relative to initiation codon
11
SLC25A38
chr3
NM_017875.2
7
100,00%
-199 relative to initiation codon
10
TPI1
chr12
NM_000365.5
7
100,00%
-65 relative to initiation codon
9

 

Table 2: List of missed bases in design

Genes
Coverage
Nr. Missing Bases
GRCh37/hg19 coordinates
Description
Missing HGMD-DM variants
GSR
98.94%
19
chr8:30585140-30585158
located central in exon 1
non
CYB5R3
97.25%
31
chr22:43045295-43045326
complete exon 1 in splice variant with 10 exons
non
PGD
98.93%
17
chr1:10478877-10478894
5' exon 11 (12 base) and flanking region
non
GAPDH
77.48%
245
chr12:6646744-6646989
5' exon 7 (240 base) and flanking region
non

The percentage of target bases that is covered at least 20 times (%Base20x) is at least 98.31% for the recommended Mapped Reads of 750.000. With this acceptance criteria five amplicons failed to yield >20 times coverage over the full amplicon length, listed in Table 3. Two published disease making mutations (DM) and 4 likely pathological mutations with ‘’some degree of doubt’’ (DM?) were missed, listed in Table 4 (Version HGMD 2015.3).

 

Table 3: Failed amplicons and their missing bases in exons and flanking intronic regions

Gene
Amplicon
%Base20x amplicon
Locations with base coverage below 20x
missing HGMD
 
 
 
chromosomal coordinates
description
 
GSR
AMPL7155931151
0%
chr8:30585159-30585467
initiation site and first 194 bases of exon 1
none
G6PD
ES7.G6PD_17_18
11%
chrX:153775085-153775261
initiation site and first 2 bp of exon 1
none
TPI1
ES7.TPI1_1
20%
chr12:6976555-6976758
initiation site and first 27 bp of exon 1
1x DM + 3x DM?
G6PD
AMPL7158818825
16%
chrX:153759784-153760063
termination site
1x DM?
GSR
AMPL7155931073
0%
chr8:30546605-30546927
exon 9 and both flanking regions
1x DM

 

Table 4: HGMD DM and DM? variants located in failed amplicons

Gene
HGMD reference
GRCh37/hg19
HGVS
Variant class
Phenotype
Reference
TPI1
CR961739
chr12:6976669
c.-62T>G
DM?
Triosephosphate isomerase deficiency
Watanabe (1996) Am J Hum Genet 58: 308
TPI1
CR993998
chr12:6976685
c.-46T>G
DM?
Triosephosphate isomerase deficiency
Watanabe (1996) Am J Hum Genet 58: 308
TPI1
CR962730
chr12:6976688
c.-43T>G
DM?
Triosephosphate isomerase deficiency
Watanabe (1996) Am J Hum Genet 58: 308
TPI1
CM962414
chr12:6976732
c.2T>A
DM
Triosephosphate isomerase deficiency
Schneider (1996) Blood Cells Mol Dis 22: 82 
G6PD
CR133485
chrX:153759858
c.*357A>G
DM?
Glucose-6-phosphate dehydrogenase deficiency
Amini (2013) J Hum Genet 58: 189 
GSR
CM074267
chr8:30546726
c.993G>A
DM
Glutathione reductase deficiency
Kamerbeek (2007) Blood 109: 3560 

 

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