Saakshi Bangera, DY Patil School of Biotechnology and Bioinformatics
Copy number variation is the phenomenon of deviation of an individual from a diploid state of its genome. Normally, humans have two copies of each locus in their genome. Any deviation from this can cause Mendelian disorders. Due to the availability of short-read sequencing technologies, several efforts have been made to characterize smaller copy number variants (CNV). Due to the data burden and a large sequencing cost, exome sequencing (ES) is the favoured clinical sequencing system. Exome sequencing mostly aims at identifying single-nucleotide variants and mutations (insertion or deletion). Existing data analysis methodologies lack the potential to detect variations at an exon-level.
Current technology adequately detects large CNVs but needs a large sample size ranging from dozens to hundreds. Due to insufficient power in exome sequencing studies and limited access to genome sequencing data, the clinical importance of CNVs at the exon level is unknown. Intragenic CNVs make up for 9.8% of pathogenic variants and 1.9% of total variants.
Target sequencing uses sequence-specific oligonucleotides to capture the desired loci. The differential efficiency of these oligonucleotides allows variable read-depth throughout the exome. The length of targeted fragments and GC content influence the observed variable read-depth. Most of the exome sequencing tools include a rectification for GC content and exon length.
Comparing multiple individually-captured samples leads to the variable read-depth problem. Independently captured samples present a large noise as compared to similarly captured samples. ExomeDepth identifies a subset of samples within other less variable samples, to avoid the variation due to individual capture.
The study demonstrates multiplexing the target across samples to reduce inter-sample variance and increase the power of detecting CNVs. The study also introduces a novel algorithm to utilize multiplexed capture data to estimate exon-level variation.
Findings of the study:
In pre-capture multiplexing, a reduction in inter-sample variance is demonstrated. This leads to an increase in the power to detect exon-level copy number variation. The study reports that the reduction in the inter-sample variance leads to proper control selection for ExomeDepth. Multiplexed capture provides appropriate controls for most samples as compared to independently-captured samples. Two platforms were compared to evaluate the inter-sample variance –
- Agilent SureSelectXT2
- Integrated DNA Technologies xGen Lockdown Probes
This study suggests that a large database provides control samples to estimate copy number variation. The simulation analysis of this study reports that both mcCNV and ExomeDepth can detect single-exon variants while keeping a slow-false discovery rate.
Conclusion:
The study recommends the following –
- Research activities adjust protocols to multiplex samples
- Before capture, check the library balance
A collection of an average of 225 filtered. The medical community lacks a piece of robust exome-wide information about the occurrence of small variants. With increased complexity, multiplexed capture is capable of revealing missed CNVs and can also increase a patient’s diagnostic efficiency. However, careful designing is important in multiplexed capture. By multiplexing the capture step, the capture pool is defined as the set of controls. This limits the need for regular reanalysis because the growing database prevents the over-selecting of samples with the same variants.
Also read: Haploflow: Tracing the disease back to its Origin
Reference:
- Filer, D.L., Kuo, F., Brandt, A.T. et al. Pre-capture multiplexing provides additional power to detect copy number variation in exome sequencing. BMC Bioinformatics 22, 374 (2021). https://doi.org/10.1186/s12859-021-04246-w
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Author info:
The author is currently pursuing MSc in Biotechnology from DY Patil School of Biotechnology and Bioinformatics. She believes that she doesn’t have a specific area of interest yet, but wishes to explore toxicology and food biotechnology. She’s quite passionate about Biotechnology and aims to grab every opportunity she comes across.
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