Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17

Abstract

Background: We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage.

Results: Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs.

Conclusion: We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS.

Figure 1

Feature map of the 8 Mb region of pig chromosome 17. Each locus is depicted according to type, orientation and position. The tiling path of the sequenced BACs is shown along the top. Below this, the distribution of repeats and C + G content is shown. Box 1 illustrates the zinc-finger locus expansion that has occurred in mouse between EDN3 and PHACTR3. The three regions described in the comparative analyses, PTPN1-CYP24A1, PFDN4-VAPB and STX16-SYCP2, are defined using double-headed arrows.

Figure 2

Comparison of GNAS transcripts in human, pig and mouse. A screenshot taken from VEGA Pig MultiContigView, comparing GNAS transcripts annotated in human (top panel), pig (middle panel) and mouse (bottom panel). The vertical blues lines joining loci in VEGA MultiContigView represent orthologous relationships between loci across species.

Figure 3

Comparison of 5× and 7.5× coverage assemblies. Dot-plots of finished BAC sequence against either 5× or 7.5× assembled sequence for BACS (a) CH242-247L10 and (b) CH242-155M9. Individual contigs, represented on the x-axis, are separated by vertical green lines. In (a) the black rectangle depicted on the graphs represents the GNAS downstream region. In (b) the black rectangle depicted on the graphs defines the vicinity of the pig C20orf106 locus.