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The RBC Membrane AmpliSeq Panel v1 (IAD89957_197) is covering 99.05% of the desired regions (all coding regions (exons), flanking intronic regions, untranslated regions and promotor areas with known (likely) pathogenic variants).: SPTA1, SPTB, ANK1, SLC4A1, EPB41, EPB42, RHAG, PIEZO1, SEC23B and UGT1A1 and include all known disease making variants listed in HGMD professional (Version HGMD 2015.3) (see Table 1 and 2)

Table 1: List of submitted genes and coverage of the design

Gene
Chromosome
NCBI Transcript
Exons
Submitted Bases
Coverage
Coverage 5' of initiation site (flanking sequence and 5'UTR)
Amplicons
ANK1
chr8
NM_001142446.1
43
3026
100,00%
204 base before initiation site
57
EPB41
chr1
NM_001166005.1
21
2806
100,00%
149 base before initiation site
20
EPB42
chr15
NM_000119.2
13
2296
100,00%
68 base before initiation site
15
PIEZO1
chr16
NM_001142864.2
51
8076
99,42%
base 34 of exon1
62
UGT1A1
chr2
NM_000463.2
5
1892
100,00%
124 base before initiation site
10
RHAG
chr6
NM_000324.2
10
1330
100,00%
95 base before initiation site
10
SEC23B
chr20
NM_032986.3
20
3455
100,00%
46 base before initiation site
28
SLC4A1
chr17
NM_000342.3
20
2926
100,00%
83 base before initiation site + regions of -3420 and -2865 before init. site
23
SPTA1
chr1
NM_003126.2
52
7780
96,85%
154 base before exon 2
52
SPTB
chr14
NM_001024858.2
35
7406
100,00%
240 base before exon 1 (incl promotor)
50

Table 2: List of missed bases in design

Genes
Coverage
Number Missing Bases
GRCh37/hg19 coordinates
Description
Missing HGMD-DM variants
ANK1
99,42%
38
chr8:41753965-41754003
flanking region and first 33 bases exon 1
non
PIEZO1
96,85%
74
chr16:88851303-88851377
both flanking regios and Exon 1 (64 bases)
non
 
 
17
chr16:88803041-88803058
flanking region and last 12 bases exon 11
non
 
 
5
chr16:88800153-88800158
flanking site (5base) of 5' exon 18
non
 
 
158
chr16:88782374-88782532
flanking region and first 153 bases exon 50
non

The percentage of target bases that is covered at least 20 times (%Base20x) is at least 99.44% for the recommended Mapped Reads of 750.000. This acceptance criteria will result in a coverage of all published disease causing variants listed in HGMD (downloaded November 2015). Two partial exon regions and their flanking intronic regions are not covered for 20 times. Both regions do not contain disease causing variants listed in HGMD professional (Version HGMD 2015.3).

Table 3: Bases possibly missed in exons and flanking intronic regions

Chr
GRCh37/hg19 coordinates
Amplicon id
Gene
Missing Bases
Exon
Missing HGMD
 
Start
End
 
 
 
 
 
chr16
88798299
88798318
AMPL7155929112
PIEZO1
20
22
non
chr16
88786834
88786940
AMPL7155930944
PIEZO1
106
41
non

Background information