This repository hosts the analysis pipeline and the relevant scripts described in the manuscript "A genome-scale CRISPR deletion screen in Chinese Hamster Ovary cells reveals essential regions of the coding and non-coding genome"_ [doi]. The work aims to uncover the genomic regions essential for CHO cell survival using a genome-scale CRISPR screening approach.
[Abstract]
./
├── config/ # (provide) configurations
├── resources/ # Raw-data and other resources
│ ├── aligned/ # Intermediate results
│ ├── flowcell_merged/ # Intermediate results
│ ├── raw/ # (provide) Raw-data
│ │ ├── 53h_dense.bed # (provide) Chromatin states****
│ │ ├── aligned_sorted_INH040x.bam # (provide) Transcripomics data
│ │ ├── reactive-genes.txt # (provide) List of reactive Genes
│ │ ├── non-reactive-genes.txt # (provide) List of non reactive Genes
│ │ ├── library_for_mageck_count.txt # (provide) pgRNA library used for MAGeCK count
│ │ ├── pgRNA-gene-id-sequence-list.fa # (provide) pgRNA library used for Bowtie2
│ │ ├── flowcell_1/ # (provide) .fasta.gz from sequencing
│ │ └── flowcell_2/ # (provide) .fasta.gz from sequencing
│ ├── table_bowtieM/ # Intermediate results
│ │ └── results/ # Intermediate results
│ └── table_mageck/ # Intermediate results
│ └── results/ # Intermediate results
├── results/ # Intermediate results
│ ├── table_bowtieM/ # Intermediate results
│ └── table_mageck/ # Intermediate results
├── results_overview/ # Results produced in the workflow
│ └── table_bowtieM/ # Results produced in the workflow
│ ├── results/ # Results produced in the workflow
│ └── table_mageck/ # Results produced in the workflow
├── workflow/ # Workflow definitions
│ ├── profiles/ # Snakemake profiles
│ ├── envs/ # Conda environments
│ ├── envs/ # Conda environments
│ ├── rules/ # Snakemake rules
│ ├── scripts/ # Scripts
│ └── Snakefile # Main Snakefile
└── README.md
- Rename and unzip files using
pigz. (01_prep_data.smk) - Merge both flowcells using
cat. (02_merge_flowcells.smk) - Merge paired end reads into an overlapping fragment using
pear. (03_merge_overlapping.smk) - Sort for read directions using
fastq-grep. (04_sort_directions.smk) - Turn reversed reads using
bioawk. (05_turn_reverse.smk) - Combine amplicons using
cat. (06_combine_amp.smk) - Trimming reads using
cutadapt. (07_trim.smk) - Alignment using
bowtie2(08_bowtei2.smk) - Change .sam to .bam using
samtools. (09_samtools.smk) - Generate counttable out of .bam-files using
mageck(10_2_mageck_count_bam.smk) - Generate counttable out of .fasta-files using
mageck(11_2_mageck_count.smk) - Remove low-count guides using
Rscript. (12_2_remove_zeros.smk) - Descriptive statistics using
Rscript. (13_2_descriptive_statistics.smk) - Prepare for ranking, create table with samples using
Rscript. (14_2_prepare_mageck_test.smk) - Restructure directory using
mv. (15_2_move_files.smk) - Guide ranking using
mageck(17_2_test_mageck_rra.smk) - Combine results from "counttable .bam" and "counttable .fasta" using
Rscript. (18_2_combine_tables.smk) - combine informatino in a table using
Rscript. (20_summary_table.smk) - Compare transcripts in essential regions using
Rscript. (21_find_transcripts.smk) - Normalisation of transcript-data using
Rscript. (22_normailse_transcripts.smk) - Plot essential regions using
Rscript. (23_plot_region_details.smk) - Restructure directory for results_overview using symlinks. (25_sort_results.smk)
- Create a summary table using
Rscript. (26_create_summary_table.smk)
- update parameters within ./config/config.yaml
- provide ./raw/53h_dense.bed
$ head 53h_dense.bed
#track name="Tp4" description="Tp4 (Emission ordered)" visibility=1 itemRgb="On"
chr10 0 21000 Repressed heterochromatin 0 . 0 21000 #3399ff
chr10 21000 27400 Quiescent/low 0 . 21000 27400 #d9d9d9
chr10 27400 47200 Repressed heterochromatin 0 . 27400 47200 #3399ff
chr10 47200 77800 Quiescent/low 0 . 47200 77800 #d9d9d9
chr10 77800 79200 Repressed heterochromatin 0 . 77800 79200 #3399ff
chr10 79200 146600 Quiescent/low 0 . 79200 146600 #d9d9d9
chr10 146600 155400 Repressed heterochromatin 0 . 146600 155400 #3399ff
chr10 155400 171000 Quiescent/low 0 . 155400 171000 #d9d9d9
chr10 171000 173000 Repressed heterochromatin 0 . 171000 173000 #3399ff
- provide ./raw/aligned_sorted_INH040x.bam
$ samtools view aligned_sorted_INH0401.bam | head
7001253F:661:CD4LWANXX:1:2313:13165:87523#ACATCCGACTGCGGAT 16 NW_023276806.1 7549 255 20M1671N33M7289N22M * 0 0 ACTAACCCTAACCCTAACCCTACCCCTCTAACCCTAAC
CCTATCCCTAACCCTCTAACCCTAACTCTAACCCTCT FFFFFFFFFFFFFFFFFFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFBFFF<FFFFFFFFF<FFFFFFFBBBBB NH:i:1 HI:i:1 AS:i:48 nM:i:8
7001253F:661:CD4LWANXX:1:2313:13165:87523#ACATCCGACTGCGGAT 16 NW_023276806.1 7549 255 20M1671N33M7289N22M * 0 0 ACTAACCCTAACCCTAACCCTACCCCTCTAACCCTAAC
CCTATCCCTAACCCTCTAACCCTAACTCTAACCCTCT FFFFFFFFFFFFFFFFFFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFBFFF<FFFFFFFFF<FFFFFFFBBBBB NH:i:1 HI:i:1 AS:i:48 nM:i:8
7001253F:661:CD4LWANXX:2:2205:17783:33201#ACATCCGACTGCGGAT 0 NW_023276806.1 29163 255 100M * 0 0 AGTGGACTGAAGATTTTAAAATAGCGGAGTGGATTAATGACACTATGACAACAG
AGAAATGGTAAATATATGAGCTGAGAGATCTGACAACCATTCACAT BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:98 nM:i:0
7001253F:661:CD4LWANXX:2:2205:17783:33201#ACATCCGACTGCGGAT 0 NW_023276806.1 29163 255 100M * 0 0 AGTGGACTGAAGATTTTAAAATAGCGGAGTGGATTAATGACACTATGACAACAG
AGAAATGGTAAATATATGAGCTGAGAGATCTGACAACCATTCACAT BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:98 nM:i:0
7001253F:661:CD4LWANXX:1:1215:19053:36813#ACATCCGACTGCGGAT 16 NW_023276806.1 29960 255 100M * 0 0 AGTTAATTATTTAAATGTTAATCATCAACATTCTCATTCTCTGAAGACTGTGAT
ACATTTATTTTATATTTCTTATTTAAAATTATTTATTTAATGCATG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBB NH:i:1 HI:i:1 AS:i:98 nM:i:0
7001253F:661:CD4LWANXX:1:1215:19053:36813#ACATCCGACTGCGGAT 16 NW_023276806.1 29960 255 100M * 0 0 AGTTAATTATTTAAATGTTAATCATCAACATTCTCATTCTCTGAAGACTGTGAT
ACATTTATTTTATATTTCTTATTTAAAATTATTTATTTAATGCATG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBB NH:i:1 HI:i:1 AS:i:98 nM:i:0
7001253F:661:CD4LWANXX:1:2205:14024:84479#ACATCCGACTGCGGAT 256 NW_023276806.1 32720 1 99M1S * 0 0 GCCTTAATTTGTCACCACCATGGGATGGGTTAGTAGAAGCCTTAACTCTAGTCT
TGATTATTCTTCTTTTGTTCATATTGGTCTTATATTGTGCTCATCA BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFB NH:i:3 HI:i:3 AS:i:97 nM:i:0
7001253F:661:CD4LWANXX:1:2205:14024:84479#ACATCCGACTGCGGAT 256 NW_023276806.1 32720 1 99M1S * 0 0 GCCTTAATTTGTCACCACCATGGGATGGGTTAGTAGAAGCCTTAACTCTAGTCT
TGATTATTCTTCTTTTGTTCATATTGGTCTTATATTGTGCTCATCA BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFB NH:i:3 HI:i:3 AS:i:97 nM:i:0
7001253F:661:CD4LWANXX:1:1101:1941:75923#ACATCCGACTGCGGAT 272 NW_023276806.1 34912 3 3S96M * 0 0 TCTTGGCAAGACACCGGCCAGGGTCCACGCTAGTTATGGGAATCCAGCAAGAAA
ACAGACAGATCAGAGAATTGCAACAGGAGAACAAAGAACTGCGAA 7///FFFB///B<//</<<///<///7<///<FBFFFFF<//F</F/F<//<B/F/FFFFB<BF<FFFB/<</FF/F/FFB</<FFFF<FFF/FBBB/B NH:i:2 HI:i:2 AS:i:90 nM:i:2
7001253F:661:CD4LWANXX:1:1101:1941:75923#ACATCCGACTGCGGAT 272 NW_023276806.1 34912 3 3S96M * 0 0 TCTTGGCAAGACACCGGCCAGGGTCCACGCTAGTTATGGGAATCCAGCAAGAAA
ACAGACAGATCAGAGAATTGCAACAGGAGAACAAAGAACTGCGAA 7///FFFB///B<//</<<///<///7<///<FBFFFFF<//F</F/F<//<B/F/FFFFB<BF<FFFB/<</FF/F/FFB</<FFFF<FFF/FBBB/B NH:i:2 HI:i:2 AS:i:90 nM:i:2
- provide ./raw/reactive-genes.txt & ./raw/non-reactive-genes.txt
$ head non-reactive-genes.txt
Rpe65
Nexn
Ifi44l
Ttll7
Dnai3
LOC100762860
LOC100762574
LOC100752806
Gbp5
LOC100750831
- provide ./raw/library_for_mageck_count.txt
$ head library_for_mageck_count.txt
>595-w10_NC_048595_1_1499500-1499519_NC_048595_1_1650461-1650480 GAAAACCTGATTGACACATG 595-w10
>595-w10_NC_048595_1_1499143-1499162_NC_048595_1_1650498-1650517 CCACCACTAGAAACAGGGAG 595-w10
>595-w10_NC_048595_1_1499243-1499262_NC_048595_1_1650893-1650912 CAGATGGTGGGACTATTGGG 595-w10
>595-w10_NC_048595_1_1499516-1499535_NC_048595_1_1650140-1650159 CATGTGGTGACCTTGGAACG 595-w10
>595-w10_NC_048595_1_1499812-1499831_NC_048595_1_1650218-1650237 CACAGGGAAAGAGCCTGGTG 595-w10
>595-w10_NC_048595_1_1499796-1499815_NC_048595_1_1650041-1650060 CTGTAGACTCTACAATCACA 595-w10
>595-w10_NC_048595_1_1499810-1499829_NC_048595_1_1650385-1650404 ATCACAGGGAAAGAGCCTGG 595-w10
>595-w10_NC_048595_1_1499368-1499387_NC_048595_1_1650079-1650098 CCACTGTGACACTCTGCCAT 595-w10
>595-w100_NC_048595_1_14999285-14999304_NC_048595_1_15150001-15150020 TGTGTGTGCACATACCCCTG 595-w100
>595-w100_NC_048595_1_14999286-14999305_NC_048595_1_15150169-15150188 GTGTGTGCACATACCCCTGA 595-w100
- provide ./raw/library_for_bowtie_count.txt
$ head pgRNA_gene_id_sequence_list.fa
>>595-w10_NC_048595_1_1499500-1499519_NC_048595_1_1650461-1650480
GAAAACCTGATTGACACATG
>>595-w10_NC_048595_1_1499143-1499162_NC_048595_1_1650498-1650517
CCACCACTAGAAACAGGGAG
>>595-w10_NC_048595_1_1499243-1499262_NC_048595_1_1650893-1650912
CAGATGGTGGGACTATTGGG
>>595-w10_NC_048595_1_1499516-1499535_NC_048595_1_1650140-1650159
CATGTGGTGACCTTGGAACG
>>595-w10_NC_048595_1_1499812-1499831_NC_048595_1_1650218-1650237
CACAGGGAAAGAGCCTGGTG