Pipeline to analyze insertion locations of construct in foreign genome
- 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
| 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 |
The directory running runner.sh must include:
- Fasta file for the current analysis construct. This could be generated from the .cm5 SnapGene file in Genomes/Constructs
- Fasta file for algae (copy or point from Genomes/Phaeodactylum_tricornutum.fna)
- Paired end sequencing data after QC and trimming (if needed)
- Bed file for endogenous regions on the construct. This should be manually made from the table above, and named as ${construct_name}.regions.bed. For example: 527.regions.bed
| 527 | 902 | 1342 | FCPA/LHCF4/CLP_promoter |
|---|---|---|---|
| 527 | 1347 | 1412 | Vac |
| 527 | 2145 | 2474 | FCPA-terminator |
| 527 | 2475 | 2719 | FCPB/LHCF4_promoter |
| 527 | 3095 | 3336 | FCPA-terminator |
Example for running runner.sh on 523 construct:
bash runner.sh 523 Phaeodactylum_tricornutum R1.fq.gz R2.fq.gz
Start by verifying insertion regions listed in TDNAscan-master/${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)