GAIIGlutaric Aciduria Type II
GAIIGlutaric Acidaemia Type II
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Because of the short read length generated by the GAII (36-base read protocol), each library was digested with BamHI, which removed all but 7 bases of the adapter sequences.
Events were totaled across samples, with Sanger-identified events not detected by the GS-FLX or the GAII considered as false negatives.
From each captured sample library, we submitted 5 [micro]g of sample for GS-FLX sequencing and 3 [micro]g for GAII sequencing.
The correlation between the GS-FLX and GAII platforms in mean exon read depth was 0.
The 454 and the GAII identified a large number of sequence variants within our targeted regions with <100% concordance, even between technical replicates (Fig.
Poor capture, inferred from the low read depth at this position, could have contributed to the absence of detection because this mutation was also missed by the GAII in one of the replicates; however, the presence of the homopolymer likely contributed to this result.
The GAII correctly identified 2 of the 3 germline mutations but, as mentioned above, failed to identify 1 germline mutation in MSH2 owing to poor read coverage.
The possibility of low-level somatic mosaicism cannot be completely ruled out; however, the lack of reproducibility between replicates and the failure of the GAII to detect these variants suggest false-positive calls as a more likely explanation.
In general, the GAII overcame poor read depth merely because of the higher number of reads generated by the instrument; however, this platform was not without its own limitations.
The GS-FLX and GAII platforms have differences in their ability to detect certain types of variants, which must be taken into account; however, as the cost and error rates of these technologies continue to decrease while the output continues to increase, we foresee their implementation for clinical diagnostics in the near future.
Acknowledgments: We acknowledge Bruce Eckloff and Yan Asmann for assistance with GAII sequencing and data analysis, respectively.