SPANDx Genomic Profiling: Verifying LTtr and K331A RNASeq Mutations

1. Set up the directory for raw data.

   #Replace "p600" with "control", "p602" with "LT", "p605" with "LTtr", and "p783" with "K331A". Please note that the RNAseq data from the LT_K331A_d8 replicates were sequenced alongside the ChIPseq batch.
   ln -s /home/jhuang/DATA/Data_Denise_LT_DNA_Bindung/Raw_Data_ChIPseq/230306_NB501882_0417_AHMVHHBGXN/2023_022_nf_denise/nf857/1_NHDF_Donor_1_p783_S1_R1_001.fastq.gz LT_K331A_d8_DonorI.fastq.gz 
   ln -s /home/jhuang/DATA/Data_Denise_LT_DNA_Bindung/Raw_Data_ChIPseq/230306_NB501882_0417_AHMVHHBGXN/2023_022_nf_denise/nf858/2_NHDF_Donor_2_p783_S2_R1_001.fastq.gz LT_K331A_d8_DonorII.fastq.gz
   mv V_8_2_4_p600_d8_DonorI.fastq.gz    control_d8_DonorI.fastq.gz
   mv V_8_2_3_p600_d8_DonorII.fastq.gz   control_d8_DonorII.fastq.gz
   mv V_8_4_2_p602_d8_DonorI.fastq.gz    LT_d8_DonorI.fastq.gz
   mv V_8_4_1_p602_d8_DonorII.fastq.gz   LT_d8_DonorII.fastq.gz
   mv V_8_2_4_p605_d8_DonorI.fastq.gz    LTtr_d8_DonorI.fastq.gz
   mv V_8_2_3_p605_d8_DonorII.fastq.gz   LTtr_d8_DonorII.fastq.gz
   

2. Execute SPANDx to produce the mapping profile. For the GenBank file used, refer to the provided link.

   LT_wt.gbk

   conda activate spandx
   [genbank copying]
   mkdir ~/anaconda3/envs/spandx/share/snpeff-4.3.1t-5/data/LT_wildtype
   cp LT_wt.gbk ~/anaconda3/envs/spandx/share/snpeff-4.3.1t-5/data/LT_wildtype/genes.gbk
   vim ~/anaconda3/envs/spandx/share/snpeff-4.3.1t-5/snpEff.config
   /home/jhuang/anaconda3/envs/spandx/bin/snpEff build -genbank LT_wildtype      -d
   
   #The mutation in LTtr is located approximately at position 780, while the K331A mutation in LT is found near position 993 out of a total of 2454 nt.
   MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPDKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPHGTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSSSGSSSREETTNSGRESSTPNGTSVPRNSSRTDGTWEDLFCDESLSSPEPPSSSEEPEEPPSSRSSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTPPKPKKNRETPVPTDFPIDLSDYLSHAVYSN<K>TVSCFAIYTTSDKAIELYDKIEKFKVDFKSRHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYKLLQENKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSRLKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKKHLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSGKTSFAAALIDLLEGKALNINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQGINNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPKANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGKFCQMIENVEAGQDPLLNILIEEEGPEETEETQDSGTFSQ*
   
   nextflow run spandx/main.nf --fastq "Raw_Data_RNAseq_K331A_d8_SPANDx/*.fastq.gz" --ref LT_wt.fasta --annotation --database LT_wildtype --pairing SE -resume
   

3. Open the BAM files created in the previous step using IGV.

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