Rat_to_Mouse

Developer: Guanqiao Feng 2019

Pipeline used in "Wang J*, Zhang T*, Johnston CJ, Kim SY, Gaffrey MJ, Chalupa D, Feng G, Qian WJ, McGraw MD, Ansong C. 2020. Protein thiol oxidation in the rat lung following e-cigarette exposure. Redox biology. 37: 101758."

This pipeline compared cysteine sites identified in rats in the current study with cysteine sites identified in mice from a previous study (Nature Communication 9,2018,1581). These cysteine sites are subjected to oxidation in response to redox stress.

It includes the following steps: data cleaning -> ortholog identification -> sequence alignment -> conserved and unique cysteine site identification.

The study 'covered 6682 unique rat cysteine sites, among which 1805 sites were also characterized in mice.'"

step 1. clean the fasta files

This step takes input files (Rattus_norvegicus_UniProt_MoTrPAC_2017-10-20.fasta & M_musculus_Uniprot_SPROT_2017-04-12.fasta) and generate output files (Rattus_norvegicus.fasta & Mus_musculus.fasta). It cleans the name, puts sequence into one line, only keeps the first gene if multiple genes with the same name exist in the file (only for Rat file).

$ perl clean_fasta_0.pl

step 2. extract Rat genes

This step extracts rat genes (E_cig_global_pep_norm_log2FC_annotated_with_Cys_sites.csv, total of 2825 genes) from input file (Rattus_norvegicus.fasta) and generates output file (Rattus_norvegicus_extract.fasta).

$ perl extract_RAT_1.pl

step 3. blast search

This step blasts Rattus_norvegicus_extract.fasta (query) against Mus_musculus.fasta (target) and only keep the best blast hit. Output file is Rat_to_Mouse_noe_list.txt.

$ makeblastdb -in Mus_musculus.fasta -dbtype prot -out Mus.db

$ blastp -db Mus.db -query Rattus_norvegicus_extract.fasta -out Rat_to_Mouse_noe.txt -outfmt 6 -max_target_seqs 1

$ cut -f 1,2 Rat_to_Mouse_noe.txt | uniq | sort -k2 > Rat_to_Mouse_noe_list.txt

$ wc -l Rat_to_Mouse_noe_list.txt
      2824
      # 2824 out of 2825 Rat genes have blast hit.

The one rat gene which do not have blast hit in mouse database is "A0A0G2K786_RAT".

step 4. extract ortholog sequences

This step extracts sequences for rat and mouse gene pairs (Rat_to_Mouse_noe_list.txt) from this two files (Rattus_norvegicus_extract.fasta & Mus_musculus.fasta). It generates 2824 files (1_genes.txt, 2_genes.txt...)

$ perl find_list_gene.pl

step 5. alignment

This step uses software muscle v3.8.1551 to do the alignment. It generate 2824 files under OUT/ directory (1_aln.txt, 2_aln.txt...)

$ makedir OUT

$ perl muscle.pl

step 6. extract cycteine position information

This step extracts cycteine position form 2824 *_aln.txt alignment files and output an excel file "cycteine_pos_summary.txt". Position indicates the position information in the respective protein sequence.

$ perl extract_cycteine.pl

$ head cycteine_pos_summary.txt
Rat_gene	Rat_pos	Mouse_gene	Mouse_pos
1433B_RAT	96	1433B_MOUSE	96
1433B_RAT	191	1433B_MOUSE	191
1433E_RAT	97	1433E_MOUSE	97
1433E_RAT	98	1433E_MOUSE	98
1433E_RAT	192	1433E_MOUSE	192
1433F_RAT	97	1433F_MOUSE	97
1433F_RAT	112	1433F_MOUSE	112
1433F_RAT	194	1433F_MOUSE	194
A0A0G2K2B8_RAT	125	1433F_MOUSE	97

$ cut -f 1 cycteine_pos_summary.txt | sort | uniq | wc -l
      2813
      # 2812 out of 2824 rat genes have cycteine position in ortholog with rat genes.

The left 12 rat genes are:

TSPO_RAT, PPIB_RAT, M0RCJ6_RAT, M0RBQ5_RAT, M0R523_RAT, H4_RAT, GSTA4_RAT, F1M0R2_RAT, D3ZE02_RAT, A0A0G2JUA5_RAT, A0A0G2JU96_RAT, A0A0G2JT56_RAT