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All deletion mutations are sequenced.
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The relatively high coverage of our assemblies (Table 1) makes unlikely that disrupting mutations are sequencing errors.
Using their predicted locations, only mutations near nucleotides that could be mutated to potential stop or splice junction mutations were sequenced.
Suspicious fragments indicating mutations were sequenced on a Licor 4000L sequencing machine.
All mutations were sequenced (Genewiz) to confirm that the DNA sequence contained only the desired point mutation.
For both samples A and B, the mutations were sequenced at a median read depth of >3,000× in all runs.
Four of the five new Cyclin E mutations were sequenced and the predicted protein changes identified (Table 1).
In order to verify the presence of the desired mutation, the ALK region corresponding to the mutations was sequenced (Fig. S1A).
Most mutations were sequenced in both directions using either the same forward or reverse primers as for PCR or an internal primer designed for sequencing.
Following DNase1 treatment and reverse transcription of the RNA, regions of complementary DNA encompassing the putative pathogenic NDUFS2 mutations were sequenced.
The conventional approach for genotyping RET mutations is sequencing the exons.
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