Here's a new preprint by @shishi.luo & co :
Genotyping Allelic and Copy Number Variation in the Immunoglobulin Heavy Chain Locus
Shishi Luo, Jane A Yu, Yun S. Song
The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV) locus, which plays an integral role in the adaptive immune response, is an example of a genomic region that is known to vary in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here, we establish a convention of representing the locus in terms of a reference panel of operationally distinguishable segments defined by hierarchical clustering. Using this reference set, we develop a pipeline that identifies copy number and allelic variation in the IGHV locus from whole-genome sequencing reads. Tests on simulated reads demonstrate that our approach is feasible and accurate for detecting the presence and absence of gene segments using reads as short as 70 bp. With reads 100 bp and longer, coverage depth can also be used to determine copy number. When applied to a family of European ancestry, our method finds new copy number variants and confirms existing variants. This study paves the way for analyzing population-level patterns of variation in the IGHV locus in larger diverse datasets and for quantitatively handling regions of copy number variation in other structurally varying and complex loci.
My favorite part of the paper is described thus in the introduction:
When we applied the pipeline to genotype the IGHV locus in a sixteen member family, we found evidence of new haplotypes that are mosaics of the existing reference haplotypes and haplotypes that might be transitional between them. We saw examples where offspring inherit structurally different haplotypes from each parent, and where high copy number variation exists within the family. We also identified a putative new allele.
Germline gene nerds like @Felix_Breden may be wondering about the cells used to generate the sequence data, which is discussed towards the end of the paper:
The Platinum Genomes data were generated from immortalized B lymphocytes. The IGHV locus in these cell types have undergone VDJ recombination. This rearrangement, which truncates the IGHV locus, confounds the correlation between read coverage depth and copy number of a gene segment. We can see this from the pipeline output, where coverage depth tends to decrease towards the centromeric (6-1 segment) end of the locus. The extent of this decrease can be quite marked, for example in the case of NA12877, or not noticeable at all, for example in NA12891 (Fig. 7A; the distribution of read coverage depth of all the individuals is summarized in S5 Fig). If one knew the number of B cell lineages used to prepare the library and the fraction of haplotypes that underwent rearrangement, it is possible to adjust the raw coverage values to reflect actual coverage values (S1 Appendix). However, in the case of the Platinum Genomes data,this information is unavailable. As whole-genome sequencing becomes more widespread, we anticipate that datasets from other cell types will become available and this issue will be resolved.