Signatures of Crested Ibis MHC Revealed by Recombination Screening and Short-Reads Assembly Strategy

<div><p>Whole-genome shotgun (WGS) sequencing has become a routine method in genome research over the past decade. However, the assembly of highly polymorphic regions in WGS projects remains a challenge, especially for large genomes. Employing BAC library constructing, PCR screening and Sanger sequencing, traditional strategy is laborious and expensive, which hampers research on polymorphic genomic regions. As one of the most highly polymorphic regions, the major histocompatibility complex (MHC) plays a central role in the adaptive immunity of all jawed vertebrates. In this study, we introduced an efficient procedure based on recombination screening and short-reads assembly. With this procedure, we constructed a high quality 488-kb region of crested ibis MHC that consists of 3 superscaffolds and contains 50 genes. Our sequence showed comparable quality (97.29% identity) to traditional Sanger assembly, while the workload was reduced almost 7 times. Comparative study revealed distinctive features of crested ibis by exhibiting the COL11A2-BLA-BLB-BRD2 cluster and presenting both ADPRH and odorant receptor (OR) gene in the MHC region. Furthermore, the conservation of the BF-TAP1-TAP2 structure in crested ibis and other vertebrate lineages is interesting in light of the hypothesis that coevolution of functionally related genes in the primordial MHC is responsible for the appearance of the antigen presentation pathways at the birth of the adaptive immune system.</p></div

Gene organization and sequence feature comparison between CSA and Chen’s report in the Sanger assembly region.

<p>The black bars indicate the scaffold in 3 sequencing strategies and the white boxes stand for gap regions. The orange blocks between the 2 scaffolds indicate the concordant sequence. The top and bottom colorized bars indicate the genes on CSA and Chen’s scaffolds, respectively.</p

Overview of recombination-based CSA strategy.

<p>(A) Fosmid library construction and target clones isolation. ~40 kb fragments of genomic DNA were ligated to the pCC2Fos vector. Then these loaded vectors were transduced into <i>E</i>. <i>coli</i> clones, which were divided into 37 pools. After screening the pools by PCR, recombinant cassettes were used to isolate target clones from positive pools. (B) Illumina library construction and sequencing. One small insert size library was constructed for each clone and a large insert size library was constructed with a mixture DNA of all clones. (C) Step by step assembly strategy of the target region. Each clone was sequenced and assembled separately to get the first class scaffold. First class scaffolds derived from the same recombinant site were assembled to second class scaffold by overlap relationship. Second class scaffolds from all sites were finally assembled to obtain the sequence of the target region.</p

Assessment of CSA assembly.

<p>(A) Alignment between CSA assembly and Sanger assembly. (B) Alignment between comparisons of the WGS assemblies with the Sanger assembly. The orange blocks indicate the consistent sequences between the 2 strategies. Single-base depth was calculated by mapping the short-read to the Sanger and CSA assemblies, respectively.</p

Gene organization and sequence features of the crested ibis MHC.

<p>(A) MHC gene map. Genes belong to the same family are indicated with the same color. Upper boxes indicate genes on a positive strand, and lower boxes indicate the opposite. The red bar indicates the MHC region that is first described in the present study. (B) Position of repetitive elements, LINEs and LTRs are represented by black bars. (C) Location of STRs. STRs with 2–5 bp repeat units are shown. (D) Location of tRNA loci. (E) Plot of local GC content in continuous 500 bp windows. The red line indicates the average GC content.</p

Assembly of the MHC region.

<p>(A) Reference assembly. Each color represents one target site. In each target site, the diamond indicates the position of the recombination cassettes; the dashed color line indicates the scaffold of each clone; the solid color line indicates the second class scaffold of each target site. The black bold line in the middle indicates the 3 super-scaffolds of the MHC. Recombinant sites C3a and C3b came from the same recombination cassette. (B) Partial haplotype separation. Paired black lines in addition to the super-scaffolds indicate the separated regions; short bars on paired black lines indicate the SNP or Indel differences between 2 chromosomes; the dashed colored lines indicate the clones used for determining the haplotypes.</p

Assembly differences between Sanger and CSA.

<p>Assembly differences between Sanger and CSA.</p