WGS

Processing WGS data with ASCAT

Although we recommend running Battenberg on WGS data to get accurate clonal and subclonal allele-specific copy-number alteration calls, ASCAT can still be used to get a fast ploidy/purity estimate. To this end, we pre-generated a set of loci, alongside with GC content and replication timing correction files.

Briefly, such a list was derived from 1000 Genomes Project SNPs (hg19, hg38 and T2T-CHM13):

• Biallelic SNPs with allele frequency higher than 0.35 and lower than 0.65 in any population were selected using BCFtools.
• Duplicated entries were removed using R.
• For hg19 and hg38, SNPs located in the ENCODE blacklisted regions were discarded. For T2T-CHM13, SNPs located in the excluderanges BED file from the Dozmorovlab were discarded.
• SNPs with noisy BAF (distant from 0/0.5/1) in normal samples (a.k.a probloci) as part of the Battenberg package were discarded (probloci_270415.txt.gz for hg19 and probloci.zip for hg38). For T2T-CHM13, a lifted over version (form GRCh38 to T2T) of the problematic loci from the Battenberg package were discarded.

Since hg38 data for the non-PAR region of chrX is not available (as of September 2021), hg38 data for the whole chrX comes from a lift-over from hg19.

GC content and replication timing correction files were then generated using scripts provided in the LogRcorrection folder.

Data availability:

• Loci files: hg19, hg38 and CHM13 (unzip and set loci.prefix="G1000_loci_hg19_chr" in ascat.prepareHTS)
• Allele files: hg19, hg38 and CHM13 (unzip and set alleles.prefix="G1000_alleles_hg19_chr" in ascat.prepareHTS)
• GC correction file: hg19, hg38 and CHM13 (unzip and set GCcontentfile="GC_G1000_hg19.txt" in ascat.correctLogR)
• Replication timing correction file: hg19 & hg38 (unzip and set replictimingfile="RT_G1000_hg19.txt" in ascat.correctLogR)

File format

Loci file

One file per chromosome, no header, the first column is the chromosome name and the second column is the position.

1	10642
1	11008
1	11012

Allele file

One file per chromosome, one header, the first column is the position and the second and third columns are reference and alternate nucleotides with the following conversion: A=1, C=2, G=3 and T=4.

position	a0	a1
10642	3	1
11008	2	3
11012	2	3

In the example above, SNP at position 10642 is G>A and SNPs at 11008 and 11012 are both C>G.

GC correction file

One single file, one header, the first column is the SNP ID, the second/third columns are chromosome/position and the other columns are GC% around SNPs with different window sizes.

	Chr	Position	25bp	50bp	100bp	200bp	500bp	1kb	2kb	5kb	10kb	20kb	50kb	100kb	200kb	500kb	1Mb
1_10642	1	10642	0.92	0.823529	0.762376	0.761194	0.722555	0.677323	0.625457	0.595799	0.590039	0.5845710.533734	0.458927	0.421891	0.425195	0.423964
1_11008	1	11008	0.72	0.745098	0.722772	0.741294	0.730539	0.705295	0.594703	0.593501	0.594541	0.5832120.534297	0.457987	0.42164	0.425088	0.423964
1_11012	1	11012	0.76	0.705882	0.742574	0.741294	0.726547	0.706294	0.595202	0.593964	0.594478	0.5831820.53433	0.457971	0.421633	0.425084	0.423964

Replication timing file

One single file, one header, the first column is the SNP ID, the second/third columns are chromosome/position and the other columns are replication timing data in different cell lines.

	Chr	Position	Bg02es	Bj	Gm06990	Gm12801	Gm12812	Gm12813	Gm12878	Helas3	Hepg2	Huvec	Imr90	K562	Mcf7	Nhek	Sknsh
1_10642	1	10642	49.509453	62.858498	52.757858	61.294971	51.757736	43.72905	48.088467	54.11837	58.062084	47.565636	68.790581	68.970825	57.467934	56.897934	60.012413
1_11008	1	11008	49.509453	62.858498	52.757858	61.294971	51.757736	43.72905	48.088467	54.11837	58.062084	47.565636	68.790581	68.970825	57.467934	56.897934	60.012413
1_11012	1	11012	49.509453	62.858498	52.757858	61.294971	51.757736	43.72905	48.088467	54.11837	58.062084	47.565636	68.790581	68.970825	57.467934	56.897934	60.012413

'chr'-based versus non 'chr'-based reference

Please note that loci files provided above are not 'chr'-based (chromosome names are '1', '2', '3', etc. and not 'chr1', 'chr2', 'chr3', etc.). If your BAMs are 'chr'-based, you will need to add 'chr' (Bash: for i in {1..22} X; do sed -i 's/^/chr/' G1000_loci_hg19_chr${i}.txt; done). ASCAT will internally remove 'chr' so the other files (allele, GC correction and RT correction) should not be modified and chrom_names (ascat.prepareHTS) should be c(1:22,'X').