Pitfalls in NGS Typing
J. Zingsem 10.5.2018
Transfusionsmedizinische und Hämostaseologische Abteilung
NGS will solve all Typing problems
?
We got the same message when changing Ø Ø
from serology to PCR from SSP/SSO PCR to SBT
… and we did not change, but add the techniques But we started the validation enthusiastic and unprejudiced Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 2 / 12
Some typing results
What‘s wrong?
Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 3 / 12
Linkage In the old days of serology DQ (now DQB1) was used to assess the probability for some DR (now DRB1) antigens where good monospecific sera were rare, e.g. DR5 vs. DR6 vs. DR8
Transfusionsmedizinische und Hämostaseologische Abteilung
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Here we found DRB1*11:04 + 15:01 but only DQB1*06 (no DQB1*03 for DRB1*11:04) DQB1*11:01 + 16:01 but only DQB1*06 (no DQB1*03 for DRB1*11:01) DRB1*07:01 + 14:01 but only DQB1*05 (no DQB1*02 for DRB1*07, but a warning)
Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 5 / 12
Not correctly marked „homozygous“ samples
Is this sample hetero- or homozygous? Let‘s see the details: Transfusionsmedizinische und Hämostaseologische Abteilung
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Homo – hetero? B*40:01:45 excluded after inspection -> homozygous
Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 7 / 12
Homozygous samples
SSP and SSO: B*13 + 49 SBT: B*13:02:01 + 49:01:01
Probable source of wrong homozygous results: SNP in the binding region of one primer
Precaution: Control of each homozygous typing using an alternative technique Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 8 / 12
Homozygous samples
- particular Situation DQB1 Ø
In 2016: 66 of 480 samples DQB1 „homozygous“
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24 of these 66 samples heterozygous in SSP + SBT Ø
In 23 of 24 false homozygous samples, a DQB1*03 allele not detected
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Consequence: Redesign of the DQB1 primers
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Result: All 24 samples heterozygous when tested using the new primers (set 3)
Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 9 / 12
Warnings
Inspection of the sequence. Transfusionsmedizinische und Hämostaseologische Abteilung
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Source of imbalance
Ø
Intron 2 of DRB1*04 has about 1200 bases more than DRB1*10.
Transfusionsmedizinische und Hämostaseologische Abteilung
10.5.2018 Zi 11 / 12
Very short résumé Ø
Many hints and warnings in Twin are helpful.
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Some warnings (homozygosity) are incomplete.
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Some warnings – describing physiological facts - could be omitted.
Ø
Software will never be ready… J
Transfusionsmedizinische und Hämostaseologische Abteilung
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