Red blood cell enzyme deficiencies NGS Panel
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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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