Widespread false gene gains caused by duplication errors in genome assemblies

Abstract

Background False duplications in genome assemblies lead to false biological conclusions. We quantified false duplications in popularly used previous genome assemblies for platypus, zebra finch, and Anna’s Hummingbird, and their new counterparts of the same species generated by the Vertebrate Genomes Project, of which the Vertebrate Genomes Project pipeline attempted to eliminate false duplications through haplotype phasing and purging. These assemblies are among the first generated by the Vertebrate Genomes Project where there was a prior chromosomal level reference assembly to compare with. Results Whole genome alignments revealed that 4 to 16% of the sequences are falsely duplicated in the previous assemblies, impacting hundreds to thousands of genes. These lead to overestimated gene family expansions. The main source of the false duplications is heterotype duplications, where the haplotype sequences were relatively more divergent than other parts of the genome leading the assembly algorithms to classify them as separate genes or genomic regions. A minor source is sequencing errors. Ancient ATP nucleotide binding gene families have a higher prevalence of false duplications compared to other gene families. Although present in a smaller proportion, we observe false duplications remaining in the Vertebrate Genomes Project assemblies that can be identified and purged. Conclusions This study highlights the need for more advanced assembly methods that better separate haplotypes and sequence errors, and the need for cautious analyses on gene gains.

False gene and chromosome losses in genome assemblies caused by GC content variation and repeats

“…Genomic regions in the VGP assemblies with no alignment to the previous assembly were used as an approximation of missing regions in the previous assembly, and vice versa. We removed the genes that were falsely duplicated due to haplotype separation errors or sequence errors in the VGP assemblies, which were identified in our companion study [ 17 ], so that they would not create false missing genes in the previous assemblies.…”

“…We found that there were some sequences in previous assemblies not present in the VGP assembly. Some of these were false haplotype duplications in the previous assembly that were correctly prevented in the primary VGP assembly (see our companion study [ 17 ]). However, we discovered a few sequences in the previous assemblies that were incorrectly missing in the VGP assemblies (Additional file 1 : Table S3).…”

“…9 b, c, ANOVA, p < 0.0001). These and other findings of some remaining false gene duplications [ 17 ] led us to reassemble the VGP male zebra finch assembly using improved algorithms (GCA_003957565.4). However, the missing 2.7 Mb sequence was again found only in the alternate assembly.…”

“…Genomic sequences from the VGP assemblies identified as missing or present in the prior assembly were extracted and concatenated to calculate GC and repeat content in each 10-kbp non-overlapping window. Because the VGP zebra finch chromosome 29 assembly included large false duplications [ 17 ] (Additional file 1 : Table S5), we merged them with BEDtools at 25 kbp maximum distance, and then excluded them for calculations of gene density, GC content, missing ratio, and repeat content.…”

“…This enabled the analysis on the climbing perch, which no annotation was available for its prior assembly. All genes found to be falsely duplicated in the VGP assemblies [ 17 ] were excluded. Using gffutils ( https://github.com/daler/gffutils ), coordinates of each region were extracted from the filtered GFF file and merged with BEDtools merge for the VGP assemblies.…”