Computational protocol: DPY-17 and MUA-3 Interact for Connective Tissue-Like Tissue Integrity in Caenorhabditis elegans: A Model for Marfan Syndrome

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Protocol publication

[…] The genomes of the mutant strains after outcrossing twice against N2 were sequenced using Illumina sequencing. Sequences were aligned to the WS220 reference C. elegans genome with bowtie2 (), variants called with the samtools/bcftools suite (), and effects on gene function predicted and variants filtered with snpEff and SnpSift (). Further specific analyses used vcftools (), bedtools (), and custom scripts.After whole genome sequencing, we found a single deletion in the mua-3 gene, but no other mutations in the range of 9.8 Mb to 10.5 Mb for chromosome III. The mutation was also validated because the original strain was from the knockout consortium of C. elegans, where the mutations are generated by the UV/TMP method to create deletions. The new allele of mua-3(uy19) contains a 488-bp in-frame deletion that includes parts of the 30th and 31st exons and the short intron between them. The flanking sequences are as follows: 5′ end of the deletion: 5′-TGAGGAGAATGGATA-3′ and 3′ end of the deletion: 5′-CACCACAGTCCAGTC-3′. The deletion results in deletion of the following 131 amino acids:RCRCRNGYHD DDPAHPGHRC SFMINECDSS NLNDCDRNAN CIDTAGGYDC ACKAPYRDEG PPQSPGRICR LNECLNPNRN TCDRNADCRD LDYGYTCTCR HGFYDQSPNP QEPGRICIEF QQEEHIERVK V. [...] The phylogenetic tree was created using the MEGA program. CLUSTALW was used for multiple sequence alignment. The p-distance method was used to calculate distance and the neighbor-joining method was used to create a tree. The tree was statistically evaluated by using the bootstrap method (1000 times), and robustness of the tree branch was indicated at the branch. The following protein sequences were used for the phylogenetic analysis: C. elegans MUA-3 (NP_001022674.1), C. elegans MUP-4 (NP_498645.1), C. elegans FBN-1 (NP_001263711.1), Drosophila melanogaster DP (Dumpy; NP001260037.1), Homo sapiens FBN1 (NP_000129.3), FBN2 (NP_001990.2), FBN3 (NP_115823.3), and EYS (Eyes shut; NP_001278938.1). H. sapiens EYS sequence was included as an outgroup member to infer the root of this unrooted tree. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, bcftools, SnpEff, SnpSift, VCFtools, BEDTools, MEGA, Clustal W
Applications Phylogenetics, WGS analysis
Organisms Caenorhabditis elegans, Homo sapiens
Diseases Marfan Syndrome
Chemicals Amino Acids