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TruncatedAnalysis

Pipeline to analyze insertion locations of construct in foreign genome

Required Programs

  • samtools 1.9
  • bedtools v2.29.2
  • Python 2.7
    • numpy==1.16.5
    • pandas==0.24.2
  • BLAST 2.9.0+
  • bedops 2.4.37
  • BWA 0.7.12
  • picard FilterSamReads 2.22.9

Endogenous regions per construct

construct FCPA/LHCF4/CLP promoter UTR Vac FCPA-terminator FCPB/LHCF4 promoter UTR FCPA-terminator
523 902-1342 1347-1412 2175-2504 2505-2749 3125-3366
527 902-1342 1347-1412 2145-2474 2475-2719 3095-3336
663 902-1342 1386-1451 2034-2363 2364-2608 2984-3225
685 899-1898 1899-1928 1929-1994 2577-2906 2907-3151 3527-3768
707 all colonies 899-1400 1463-1528 2261-2590 2591-2835 3211-3452
730 899-1898 1899-1915 1937-2002 2441-2770 2771-3015 3391-3632
833 1211-2210 2211-2240 2242-2307 2882-3211 3224-4223 4224-4253 5738-5979

How to run analysis

The directory running runner.sh must include:

  1. Fasta file for the current analysis construct. This could be generated from the .cm5 SnapGene file in Genomes/Constructs
  2. Fasta file for algae (copy or point from Genomes/Phaeodactylum_tricornutum.fna)
  3. Paired end sequencing data after QC and trimming (if needed)
  4. Bed file for endogenous regions on the construct. This should be manually made from the table above, and named as ${construct_name}.regions.bed. For example: 527.regions.bed
527 902 1342 FCPA/LHCF4/CLP_promoter
527 1347 1412 Vac
527 2145 2474 FCPA-terminator
527 2475 2719 FCPB/LHCF4_promoter
527 3095 3336 FCPA-terminator

Example for running runner.sh on 523 construct: 

bash runner.sh 523 Phaeodactylum_tricornutum R1.fq.gz R2.fq.gz

Results

Start by verifying insertion regions listed in TDNAscan-master/${construct_name}tdnascan/5.${construct_name}tdnascan_insertion.bed do not fall on endogenous regions

Manually go over results in TDNAscan-master/${construct_name}tdnascan/3.${construct_name}tdnascan_informativeGenome_IR.txt and identify regions of possible insertion by comparing the reads aligned on the algae to the reads aligned to the construct. This could be achieved by running extractRegion.sh to produce bam file aligned to the construct for reads originated in specific region on the algae genome for example:

bash extractRegion.sh NC_011672.1-741709 TDNAscan-master/${construct_name}tdnascan/1.TDNA_sort.bam TDNAscan-master/${construct_name}tdnascan/3.${construct_name}tdnascan_informativeGenome_sort.bam

For each insertion identify if gene of interest is present. The genes can be found in Genomes/Constructs/probes

Compare number of insertions with the number of copies identified in the Southern Blot experiment (southernBlot.txt)