question from Ping Wu

[Ping Wu]

Hi Anton,

We want to use QuEST to analyse the ATAC-seq result. I generated a chicken galgal4 genome table (see attachment) and an ATAC-seq bam file and an Input bam file. I understood that QuEST may not recognize bam files. So I convert the bam file to bed (and Sam file),

In my ATAC_seq_test2 folder, I have ATAC1.bed, Input.bed, and galGal4_genome_table.txt

When I type the following command:

~/Desktop/QuEST_2.4/generate_QuEST_parameters.pl -solexa_align_ChIP ./ ATAC1.bed -solexa_align_RX_noIP ./Input.bed -gt ./galGal4_genome_table.txt -ap ./QuEST_analysis -ChIP_name ATAC

It shows: Warning: unknown parameter: ATAC1
or no contig in the genome table.

May I know the possible problem? Thanks

Ping

[Hunter Richards]

If you copy-pasted the command, you have a space in between "./" and "ATAC1.bed"

./ ATAC1.bed

     ^

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Hunter Richards

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[Anton]

Hi Ping,

also, you need to use -sam_align_ChIP flag and provide .sam files (e.g. with samtools).

.bed files right now cannot used as input QuEST.

Anton

[Ping Wu]

This time I used sam files in the folder ATAC_seq_test3 and changed the flag:

~/Desktop/QuEST_2.4/generate_QuEST_parameters.pl -sam_align_ChIP ./ ATAC1.sam -sam_align_noIP ./ Input.sam -gt ./galGal4_genome_table.txt -ap ./QuEST_analysis -ChIP_name ATAC

but it shows:
Warning: unknown parameter: ATAC1
pw@pw-XPS-8700:~/Desktop/ATAC_seq_test3$

On Thursday, May 28, 2015 at 10:04:41 AM UTC-7, Ping Wu wrote:

[Tara J Nulton]

Try removing the spaces in front of ATAC1 and Input.sam as suggested earlier...  Tara

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Tara Letts | Technology Manager

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[Ping Wu]

Thanks. Tara.
I removed the spaces. But it shows:

Warning: unknown parameter: -sam_align_noIP
pw@pw-XPS-8700:~/Desktop/ATAC_seq_test3$

[Hunter Richards]

the flags are -sam_align_ChIP and -sam_align_RX_noIP

[Ping Wu]

Thanks, Hunter.

Now I use the command:
~/Desktop/QuEST_2.4/generate_QuEST_parameters.pl -sam_align_ChIP ./ATAC1 -sam_align_RX_noIP ./Input -gt ./galGal4_genome_table.txt -ap ./QuEST_analysis -ChIP_name ATAC

It shows:
Found the following contigs in the genome table:
There were not contigs found. Please correct reference path or genome table.

Ping

[Anton]

Is it tab-delimited?

You can try generating the table with QuEST by providing fasta files with just these contigs. Probably it's easiest to start with just fully assembled chromosomes (chr1 - chr25 and chrW). 

[Hunter Richards]

I agree with Anton. I have a directory called Reference_Genomes and in it I have mm9, mm10, hg18, hg19, etc. (fasta files) in their own directories. I just use the path to the genome assembly as needed when running QuEST (with the -ap flag). The mm9 genome for instance is only about 2.5GB. So they won't take up much space. I've found I use the fasta files for many other things that require sequence, and not just the size of the chromosomes.

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[Ping Wu]

Hi Anton,

When I rebuilt the genome table by replacing the \t with a space, it works now. Thanks,

Ping