CIRCLE-seq is a tool for analysis of circularization for in vitro reporting of cleavage effects by sequencing. It is a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRIScrisPR–Cas9 genome-wide off-target mutations. It can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLEcircle-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles.

References:

• Shengdar Q Tsai, Nhu T Nguyen, Jose Malagon-Lopez, Ved V Topkar, Martin J Aryee & J Keith Joung
  CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRIScrisPR–Cas9 nuclease off-targets.
  Nature Methods, VOL.14, NO.6, p.607-614.

• CIRCLE-seq GitHub page

• Module Name: circleseq (see the modules page for more information)
• Unusual environment variables set
  • CS_HOME  installation directory
  • CS_BIN    executables directory
  • CS_SRC    source code directory
  • CS_DATA    sample input data directory

[user@biowulf]$ sinteractive 
[user@cn0861 ~]$ module load circleseq   
[+] Loading singularity  3.10.5  on cn4189
[+] Loading circleseq  1.1
user@cn0861 ~]$ mkdir -p /data/$USER/circleseq && cd /data/$USER/circleseq 

[user@biowulf]$ cp $CS_DATA/* .  
[user@biowulf]$ cp $CS_SRC/manifest.yaml . 

[user@biowulf]$ circleseq.py all --manifest ./manifest.yaml 
[08/24 08:36:38AM][INFO][circleseq] Loading manifest...
/opt/conda/envs/circleseq/circleseq-1.1/circleseq/circleseq.py:39: YAMLLoadWarning: calling yaml.load() without Loader=... is deprecated, as the default Loader is unsafe. Please read https://msg.pyyaml.org/load for full details.
  manifest_data = yaml.load(f)
[08/24 08:36:38AM][INFO][circleseq] Merging reads...
[08/24 08:36:39AM][INFO][alignReads] BWA genome index found.
[08/24 08:36:39AM][INFO][alignReads] Running paired end mapping for U2OS_exp1_VEGFA_site_1
[08/24 08:36:39AM][INFO][alignReads] bwa mem /data/user/circleseq/CIRCLEseq_test_genome.fa /data/user/circleseq/fastq/U2OS_exp1_VEGFA_site_1_merged.fastq.gz  > /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1.sam
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 2439 sequences (736578 bp)...
[M::mem_process_seqs] Processed 2439 reads in 0.356 CPU sec, 0.357 real sec
[main] Version: 0.7.17-r1188
[main] CMD: bwa mem /data/user/circleseq/CIRCLEseq_test_genome.fa /data/user/circleseq/fastq/U2OS_exp1_VEGFA_site_1_merged.fastq.gz
[main] Real time: 0.377 sec; CPU: 0.377 sec
[08/24 08:36:40AM][INFO][alignReads] Paired end mapping for U2OS_exp1_VEGFA_site_1 completed.
[08/24 08:36:40AM][INFO][alignReads] samtools sort -o /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1.bam /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1.sam
[08/24 08:36:40AM][INFO][alignReads] Sorting by coordinate position for U2OS_exp1_VEGFA_site_1 complete.
[08/24 08:36:40AM][INFO][alignReads] samtools index /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1.bam
[08/24 08:36:40AM][INFO][alignReads] Indexing for U2OS_exp1_VEGFA_site_1 complete.
[08/24 08:36:40AM][INFO][alignReads] samtools sort -o /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1_sorted.bam -n /data/user/circleseq/aligned/U2OS_exp1_VEGFA_site_1.bam
[08/24 08:36:40AM][INFO][alignReads] Sorting for U2OS_exp1_VEGFA_site_1 by name complete.
[08/24 08:36:40AM][INFO][alignReads] BWA genome index found.
[08/24 08:36:40AM][INFO][alignReads] Running paired end mapping for control_U2OS_exp1_VEGFA_site_1
[08/24 08:36:40AM][INFO][alignReads] bwa mem /data/user/circleseq/CIRCLEseq_test_genome.fa /data/user/circleseq/fastq/control_U2OS_exp1_VEGFA_site_1_merged.fastq.gz  > /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1.sam
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 967 sequences (292034 bp)...
[M::mem_process_seqs] Processed 967 reads in 0.146 CPU sec, 0.146 real sec
[main] Version: 0.7.17-r1188
[main] CMD: bwa mem /data/user/circleseq/CIRCLEseq_test_genome.fa /data/user/circleseq/fastq/control_U2OS_exp1_VEGFA_site_1_merged.fastq.gz
[main] Real time: 0.157 sec; CPU: 0.154 sec
[08/24 08:36:40AM][INFO][alignReads] Paired end mapping for control_U2OS_exp1_VEGFA_site_1 completed.
[08/24 08:36:40AM][INFO][alignReads] samtools sort -o /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1.bam /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1.sam
[08/24 08:36:40AM][INFO][alignReads] Sorting by coordinate position for control_U2OS_exp1_VEGFA_site_1 complete.
[08/24 08:36:40AM][INFO][alignReads] samtools index /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1.bam
[08/24 08:36:40AM][INFO][alignReads] Indexing for control_U2OS_exp1_VEGFA_site_1 complete.
[08/24 08:36:40AM][INFO][alignReads] samtools sort -o /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1_sorted.bam -n /data/user/circleseq/aligned/control_U2OS_exp1_VEGFA_site_1.bam
[08/24 08:36:41AM][INFO][alignReads] Sorting for control_U2OS_exp1_VEGFA_site_1 by name complete.
[08/24 08:36:41AM][INFO][circleseq] Finished merging and aligning reads.
[08/24 08:36:41AM][INFO][circleseq] Identifying off-target cleavage sites.
[08/24 08:36:41AM][INFO][circleseq] Window: 3, MAPQ: 50, Gap: 3, Start 1, Mismatches 6, Search_Radius 20
Writing counts to /data/user/circleseq/identified/U2OS_exp1_VEGFA_site_1_count.txt
Tabulate nuclease merged start positions.
Tabulate control merged start positions.
[0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0] [0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.0]
Writing matched table
Writing unmatched table
[08/24 08:36:41AM][INFO][circleseq] Visualizing off-target sites
[08/24 08:36:41AM][INFO][circleseq] Finished visualizing off-target sites
[user@cn0861 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226