stepStone v1.2

A pipeline for identification of chromothripsis breakpoints and cancer rearrangements

Download and Compile:

$ git clone  https://github.com/wtsi-hpag/stepStone.git 
$ cd stepStone 
$ bash install.sh

If everything compiled successfully you must see the final comment: "Congrats: installation successful!"

External packages

The genome aligner BWA-mem2 (https://github.com/bwa-mem2/bwa-mem2), minimap2 (https://github.com/lh3/minimap2) and samtools (http://www.htslib.org) are downloaded and compiled by stepStone.

Run the pipelines

Program: stepStone - a pipeline to identify chromothripsis breakpoints and rearrangements Version: 2.0

       $ /full/path/to/stepStone/src/stepStone command [options]           \

Commands:
-- align Align reads to a reference
-- plot Plot depth of coverage
-- breakpoint Detect breakpoints \

Read Alignments

       $ /full/path/to/stepStone/src/stepStone align           \

===Align reads to a reference for all data types: \
===Output a coordinate sorted bam file: \

       $ ./stepStone align -nodes 60 -data ccs -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
       $ ./stepStone align -nodes 60 -data ont -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
       $ ./stepStone align -nodes 60 -data ont-NLR -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
       $ ./stepStone align -nodes 60 -data ngs-HiC -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
       $ ./stepStone align -nodes 60 -data ngs-10X -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
       $ ./stepStone align -nodes 60 -data ngs-SSR -reads input_long.fasta/q <Input_Reference> <Output_sorted_bam>            \
	-nodes    60      - Number of CPUs requested                                                                       \
	-data     ccs     - PacBio HiFi                                                                      \
	-data     ont     - Oxford Nanopore Q20 or Q30                                                                      \
	-data     ont-NLR - Oxford Nanopore normal long reads (NLR)                                                         \
	-data     ngs-HiC - Hi-C reads                                                                                      \
	-data     ngs-10X - 10X reads                                                                                      \
	-data     ngs-SSR - Standard short reads such as Illumina data                                                      \

Breakpoint Detection

       $ /full/path/to/stepStone/src/stepStone breakpoint                                     \

===Detect breakpoints with aligned, and name sorted sam,bam or cram files: \

       $ ./stepStone breakpoint -data ccs -bam mysorted.bam <output_breakpoints.vcf>          \
       $ ./stepStone breakpoint -data ont -bam mysorted.bam <output_breakpoints.vcf>          \
       $ ./stepStone breakpoint -data ngs-HiC -bam mysorted.bam <output_breakpoints.vcf>      \
       $ ./stepStone breakpoint -data ngs-10x -bam mysorted.bam <output_breakpoints.vcf>      \
       $ ./stepStone breakpoint -data ngs-SSR -bam mysorted.bam <output_breakpoints.vcf>      \
	-data     ccs     - PacBio HiFi                                                   \
	-data     ont     - Oxford Nanopore Q20 or Q30                                    \
	-data     ngs-HiC - Hi-C reads                                                    \
	-data     ngs-10X - 10X reads                                                     \
	-data     ngs-SSR - Standard short reads such as Illumina data                    \
--- Note: the sam/bam/cram file has to be Name sorted! ---                                \
--- If not read name sorted, please do the following:  ---                                \
--- samtools sort -@ 60 -n your.bam new.bam ---                                           \

===Detect breakpoints with fasta/fastq long read files: \

       $ /full/path/to/stepStone/src/stepStone breakpoint                                     \
      -nodes 60 -data ccs/ont -reads input_long.fasta/q <Input_Reference> <breakpoints.vcf>   \

Coverage/Aneuploidy Plots

       $ /full/path/to/stepStone/src/stepStone plot           				\

===Plot depth of coverage for all data types:
====Input a coordinate sorted bam file
====Output a tmp directory containing coverage images for 23 chromosomes chr{1,22,X} \

       $ /full/path/to/stepStone/src/stepStone plot -bam mysorted.bam -sample cancer-XXX    \
         -hight 180 -window 100 -denoise 1  					      	\	
      			 								\
      -sample:   Sample name		  						\
      -hight 	(180): 	Maximum value in Y axis (read depth)  				\
      -window 	(100): 	Window size to display chromosome coordinates  			\
      -chrnum 	(23):  	Number of chromosomes  			 		\
      -denoise 	(3):  	Noise reduction option {0,1,2,3}  			\
       	  (0)  	No noise reduction option  			 		\
       	  (1)  	Average the data points after filtering high and low points within the window size	\
       	  (2)  	Fit the datasets into copy numbers after filtering/smoothing	\
       	  (3)   Dimensionless copy numbers in logscale after filtering/smoothing\

===Without noise reduction: \

       $ /full/path/to/stepStone/src/stepStone plot -bam mysorted.bam -sample cancer -denoise 0 \

Best Practices

1. Align the reads to obtain a coordinate sorted bam file;
2. Generate coverage plots;
3. samtools sort -@ 60 -n your.bam new.bam;
4. Detect breakpoints using new.bam

Note:

1. The pipeline only accepts correct chromosome names in the reference assembly, such as

   1,2,3,..., X,Y or chr1,chr2,chr3, ..., chrX, chrY

2. When you have high read coverage using -denoise {1,2}, you need to adjust "hight"; no need for -denoise 3.
3. Window size gives you a chance to display the density of data points in plots.

Please contact Zemin Ning ( [email protected] ) for any further information.