Ask the use of some of the problems encountered

[clsteam]

Dear Stephane,
pHASER has been quite helpful for me. However, I'm trying to assess allele-specific gene expression in single-cell RNA-Seq data from human lung cancer cells and I have tried it many times,But all failed(aCount=0 and bCount=0),is there a problem with my vcf file?

vcf file:

chr1    89923   .       A       T       68      .       DP=3;VDB=4.340713e-02;AF1=1;AC1=2;DP4=0,0,2,1;MQ=50;FQ=-36      GT:PL:GQ        1/1:100,9,0:16
chr1    90311   .       T       C       8.64    .       DP=17;VDB=6.089532e-02;RPB=-2.152553e+00;AF1=0.5;AC1=1;DP4=6,7,1,2;MQ=48;FQ=11.3;PV4=1,0.15,1, 0.059     GT:PL:GQ        0/1:38,0,229:40
chr1    134223  .       G       C       87      .       DP=5;VDB=2.649457e-02;RPB=8.293682e-01;AF1=0.5013;AC1=1;DP4=1,0,3,1;MQ=50;FQ=-5.45;PV4=1,0.42,1,1       GT:PL:GQ        0/1:117,0,23:26
chr1    134667  .       A       G       158     .       DP=6;VDB=7.655903e-02;AF1=1;AC1=2;DP4=0,0,3,3;MQ=50;FQ=-45      GT:PL:GQ        1/1:191,18,0:33

haplotypic_counts.txt:

- chr15   60422193        60422224        chr15_60422193_T_C,chr15_60422224_T_C   2       C,C     T,T     0       0       0       0/1     0.5
- chr15   29719135        29719136        chr15_29719135_A_G,chr15_29719136_C_T   2       G,T     A,C     0       0       0       0/1     0.5
- chr15   85748317        85748331        chr15_85748317_T_G,chr15_85748331_G_A   2       T,A     G,G     0       0       0       0/1     0.5
- chr15   84202077        84202144        chr15_84202077_C_G,chr15_84202144_G_A   2       C,G     G,A     0       0       0       0/1     0.5

python phaser.py --pass_only 0 --vcf var.flt.vcf.gz --bam $bam --paired_end 1 --mapq 10 --baseq 10 --sample $bam --blacklist hg19_hla.bed --haplo_count_blacklist hg19_haplo_count_blacklist.bed --threads 6 --o ase

I do not know much about it in this respect, can you tell me where there is a problem?
thanks

[secastel]

Hi there, yes something appears to be wrong. In particular, since it looks like read backed haplotypes are being generated (which means it has recorded the presence of those reads), but the counts are listed as 0 in the haplotypes file...

Can you please post the full output log as a result of you running the command above?

[clsteam]

Thank you for your response. My bam file are processed by Tophat2,and I do not know if this has any effect on the steps behind. Then I have attached the full output log: log.txt
As the log says, I changed the parameter --gw_phase_vcf to 1. However,the counts are still listed as 0 in the haplotypes file, the contents of output log have not changed.
input:
vcf
Other output files:
allele_config.txt
allelic_counts.txt
haplotypes.txt
haplotypic_counts.txt
variant_connections.txt

I do not know how to do this in such cases, please forgive my ignorance.

thanks,

[secastel]

Thank you for posting the logs. It appears that everything except the haplotypic_counts file was generated successfully, so there may be some sort of bug with phASER. Would it be possible for you to make available to me at least the VCF and BAM for one chromosome so that I can try to reproduce the bug on my end?

[clsteam]

There's my file here (Include only one chromsome 18):vcf and bam

thanks,

[secastel]

Hi there, unfortunately the link above does not seem to work.

[clsteam]

sorry, it can not work for some reason.
There are new links here:
vcf and bam

Thanks,

[secastel]

I identified a bug that was occurring when the --haplo_count_blacklist argument was specified. The latest version of phASER (v0.9.9.4) should address this problem. Please try again using the updated version of phASER and let me know if you are now getting haplotypic counts. Please note, that you will also need to redownload the "hg19_haplo_count_blacklist.bed" file.

[clsteam]

Thank you for upgrading the phaser, i‘ve got Generate haplotypic counts. However, it seems a bit out of trouble when i run phaser_gene_ae to generate haplotype expression quantifications, the result is all zero.

[secastel]

Hi there, it looks like the issue is that you have named your chromosomes e.g. "chr1", and the annotation files that are provided with phASER for convenience are named "1". I have updated all of the documentation links to include both annotation files with and without "chr" in the chromosome names, but here are the files you will need. Please re-run phASER and phaser_gene_ae with these files, and let me know if it worked.

ftp://ftp.nygenome.org/sec/phaser/hg19_hla.chr.bed.gz
ftp://ftp.nygenome.org/sec/phaser/hg19_haplo_count_blacklist.chr.bed.gz
ftp://ftp.nygenome.org/sec/phaser/hg19_ensembl.chr.bed.gz

[x811zou]

@secastel Hi, could you share these files with me? I am using Tophat2 and encountering some problems as well. Thank you!

[secastel]

@x811zou you can find all of the needed files (with chr in the name) on the blog post / tutorial here: https://stephanecastel.wordpress.com/2017/02/15/how-to-generate-ase-data-with-phaser/