High‐throughput complement component 4 genomic sequence analysis with C4Investigator

Abstract

The complement component 4 gene loci, composed of the C4A and C4B genes and located on chromosome 6, encodes for complement component 4 (C4) proteins, a key intermediate in the classical and lectin pathways of the complement system. The complement system is an important modulator of immune system activity and is also involved in the clearance of immune complexes and cellular debris. C4A and C4B gene loci exhibit copy number variation, with each composite gene varying between 0 and 5 copies per haplotype. C4A and C4B genes also vary in size depending on the presence of the human endogenous retrovirus (HERV) in intron 9, denoted by C4(L) for long‐form and C4(S) for short‐form, which affects expression and is found in both C4A and C4B. Additionally, human blood group antigens Rodgers and Chido are located on the C4 protein, with the Rodger epitope generally found on C4A protein, and the Chido epitope generally found on C4B protein. C4A and C4B copy number variation has been implicated in numerous autoimmune and pathogenic diseases. Despite the central role of C4 in immune function and regulation, high‐throughput genomic sequence analysis of C4A and C4B variants has been impeded by the high degree of sequence similarity and complex genetic variation exhibited by these genes. To investigate C4 variation using genomic sequencing data, we have developed a novel bioinformatic pipeline for comprehensive, high‐throughput characterization of human C4A and C4B sequences from short‐read sequencing data, named C4Investigator. Using paired‐end targeted or whole genome sequence data as input, C4Investigator determines the overall gene copy numbers, as well as C4A, C4B, C4(Rodger), C4(Ch), C4(L), and C4(S). Additionally, C4Ivestigator reports the full overall C4A and C4B aligned sequence, enabling nucleotide level analysis. To demonstrate the utility of this workflow we have analyzed C4A and C4B variation in the 1000 Genomes Project Data set, showing that these genes are highly poly‐allelic with many variants that have the potential to impact C4 protein function.

MHConstructor: A high-throughput, haplotype-informed solution to the MHC assembly challenge

“…As evidenced by the dotplot visualization showing assembly alignment gaps corresponding to the position of C4A and C4B, de novo short read assembly is not capable of discerning between C4A and C4B and their duplicates. To avoid this problem, we have implemented C4Investigator (Marin et al 2024) annotations, which designate the presence of C4A and C4B, long and short forms, and copy numbers. Another limitation associated with short read assembly of repeat content is the proper boundary designation and copy number identification of interspersed repeat elements.…”

“…In this way, we determine the HLA class II haplotype(s) for each individual. - Complement component 4 (C4A and C4B genotyping. C4A and C4B genotypes and copy numbers are assigned using C4Investigator (Marin et al . 2024) with the standard parameter values, calculating C4 copy numbers (C4A, C4B, C4S, C4L).…”

“…We here present MHConstructor, a Singularity container (Kurtzer et al . 2017) which houses an integrated suite of tools aimed at producing high-quality MHC sequence at high-throughput: A de novo assembly pipeline (Lischer and Shimizu 2017) custom adapted for application to the extended 5Mb MHC region, a short read quality control filter (Lischer and Shimizu 2017), C4Investigator for complement component 4 (C4) genotyping and copy number variation calling (Marin et al . 2024), T1K for first field DRB1 genotyping (Song et al .…”

“…These challenges have resulted in limited capacity to reliably generate MHC sequences and thoroughly query the functional role of MHC variation in disease association contexts. We here present MHConstructor, a Singularity container (Kurtzer et al 2017) which houses an integrated suite of tools aimed at producing high-quality MHC sequence at high-throughput: A de novo assembly pipeline (Lischer and Shimizu 2017) custom adapted for application to the extended 5Mb MHC region, a short read quality control filter (Lischer and Shimizu 2017), C4Investigator for complement component 4 (C4) genotyping and copy number variation calling (Marin et al 2024), T1K for first field DRB1 genotyping (Song et al 2023), a batch short read data extraction that interfaces with the 1000GenomesProject database (Auton et al 2015), and all necessary supplementary software dependencies, in a user-friendly, containerized interface.…”