Abstract 387P
Background
1p/19q co-deletion is an important diagnosis and prognosis biomarker of glioma. Fluorescence in situ hybridization (FISH), as the golden standard method, is often unachievable for sample preparation difficulties. In addition, the accuracy is limited because only a few specific genomic loci are detected to represent the status of whole chromosomal arms. Here we propose a sequencing-based method that combines coverage depth and allele frequency of heterozygous SNVs, to improve the detection of 1p/19q co-deletion.
Methods
We designed a DNA capture kit, which covers the whole 1p and 19q chromosomal arms with well-distributed probes, about one per 500kb region. Fifty-four negative and 20 positive samples of 1p/19q co-deletion, all confirmed by FISH, were involved to establish the method. DNA libraries of the samples were captured by the kit, and then sequenced on Illumina platform. After mapping the sequenced reads to the human reference genome, cumulative Stouffer's Z-scores of sequencing depth at all probe loci were calculated and compared between positive samples and negative control samples, and allele frequencies of all heterozygous SNVs on the two chromosomal arms were calculated. The 1p/19q deletion was defined as that the Z-score exceeds the cutoff and the allele frequency distribution deviates from theoretical heterozygous allele frequency 50%.
Results
Without combining information of heterozygous SNV, our method generated comparable results as other popular tools, such as CNVkit. We got 100% sensitivity and 98.1% specificity on 1p-arm and 100% sensitivity and 96.2% specificity on 19q-arm. By manually checking the false-positive cases, we confirmed that their allele frequency distribution of heterozygous SNVs deviated from 50%, indicating that the false LOH result should be caused by the fluctuation of sequencing. By combining heterozygous SNV allele frequency, the specificity of 1p and 19q detection was increased to 100% and 98%.
Conclusions
We developed a new method for detection of 1p/19q deletion with high accuracy, by improving both the capture kit design and bioinformatics algorithm to incorporate both sequencing depth and allele frequency of heterozygous SNVs.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.