A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3

Abstract

We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w(1118); iso-2; iso-3 strain and the reference y(1); cn(1) bw(1) sp(1) strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5'UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5' and 3' UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory.

Figure 1. Classification of SNPs in w¹¹¹⁸; iso-2; iso-3. The number of NSPs in each class is shown above the bar. The quality score was arbitrarily set at 70 and above for this graph.

Figure 2. Analysis of Eip63E start-gained SNP in w¹¹¹⁸; iso-2; iso-3. (A) Location of the start-gained SNP at the Eip63E locus. Notice that the reading frame is the same as the normal translation start site (TSS). (B) Conservation of 60 amino acid N-terminal region of Eip63E in w¹¹¹⁸; iso-2; iso-3 with Drosophila yakuba orthologous gene. The other sequenced Drosophila species do not have this N-terminal sequence (not shown).

Figure 3. Oc/Otd has two stop-gained SNPs in w¹¹¹⁸; iso-2; iso-3. (A) Location of the two stop gained SNPs in oc/otd. (B) Protein BLAST of Oc/Otd against the non-redundant (nr) protein database shows that only the 60 amino Hox domain flanking amino acid 100 is conserved from Drosophila to humans. The color coding shows the alignment scores.

Figure 4. CG34326 has one stop-gained SNP in w¹¹¹⁸; iso-2; iso-3 in the non-conserved C-terminal region. (A) Protein BLAST of CG34326 against the non-redundant (nr) protein database shows that only the 38 N-terminal amino acids are conserved among Drosophila species and not beyond Drosophila. The colored lines represent the homologs from the following organisms: Drosophila melanogaster, Drosophila grimshawi, Drosophila yakuba, Drosophila erecta, Drosophila virilus, Ixodes scapularis, Ixodes scapularis, and Nycticebus coucang. (B) Aligment of Drosophila melanogaster CG34326 with orthologous gene from Drosophila grimshawi. (C) Aligment of Drosophila melanogaster CG34326 with orthologous gene from Drosophila yakuba.

Figure 5. CG13958 has a stop lost SNP in w¹¹¹⁸; iso-2; iso-3. The top comparison shows the alignment of the Drosophila melanogaster reference genome with w¹¹¹⁸; iso-2; iso-3. Notice that the stop lost causes an extension of nine amino acids. The second through sixth comparisons shows the alignment of Drosophila simulans, Drosophila erecta, Drosophila yakuba, Drosophila mojavensis, and Drosophila pseudoobscura pseudoobscura (Sbjct) with the Drosophila melanogaster reference genome (Dm-ref). The number of terminal amino acids missing or gained is shown (-1 to +3).

Figure 6. Nonsynonymous to synonymous ratios along the chromosome arms in w¹¹¹⁸; iso-2; iso-3. (A) Left, Nonsynonymous SNPs at 1 Mbp intervals along the 2L chromosome arm (black) and synonymous SNPs (gray). Right, N/S ratios (NS/Syn) along the chromosome arms. Notice that N/S ratios are higher near the centromere and telomere (see text). (B-F) as in (A), but for chromosome arms 2R, 3L, 3R, 4 and X.