Question: Picard error: Sequences at index 0 don't match
0
gravatar for zachariasawaged
10 months ago by
zachariasawaged0 wrote:

I am having an issue with Picard when I try to use the CollectAlignmentSummaryMetrics tool.

So I aligned my Fastq into a BAM using Star and used a pre-made Star index from this directory: http://labshare.cshl.edu/shares/gingeraslab/www.data/dobin/STAR/STARgenomes/UCSC/hg19_inclHap_noAnnotations_SAsparseD3/

And then I am using these chromosomes.fa files as my reference for Picard: http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STARgenomes/UCSC/hg19_noAnnotations/FASTA/

I used this code to put the chr.fa files together into one fasta file and make a dict:

tar -zxvf chromFa.tar.gz
cat chr*.fa > hg19.fa
module load gatk
gatk CreateSequenceDictionary -R hg19.fa

Then when I run Picard, here is the error:

Exception in thread "main" htsjdk.samtools.util.SequenceUtil$SequenceListsDifferException: Sequences at index 0 don't match: 0/249250621/chr1 0/135534747/chr10/M5=988c28e000e84c26d552359af1ea2e1d/UR=file:/gpfs/scratch/sawagz01/hg19.fa

Any advice? Is there an issue with using two HG19 sequences from two different directories?

ADD COMMENTlink modified 10 months ago by swbarnes27.4k • written 10 months ago by zachariasawaged0
1

then when I run Picard

which tool ? which parameters ??

enter image description here

most probably you merged the fasta sequence

cat chr*.fa > hg19.fa

with an order that is not the same as the one used by a bam or a vcf file.

ADD REPLYlink modified 10 months ago • written 10 months ago by Pierre Lindenbaum126k

Hey Pierre - I mentioned it in my fist line: CollectAlignmentSummaryMetrics tool. How do I adjust the order?

ADD REPLYlink written 10 months ago by zachariasawaged0
0
gravatar for swbarnes2
10 months ago by
swbarnes27.4k
United States
swbarnes27.4k wrote:

Are you completely sure that your pre-made index was made using the chromosome.fa that you are giving to Picard?

ADD COMMENTlink written 10 months ago by swbarnes27.4k

That's my main concern. They were both from UCSC but not from the same file. I downloaded the index pre-made, and used a fa file that was in a different directory than my index.

I ended up realigning to an fa file that was in the same directory, but that gave me a new error: Sequence dictionaries are not the same size (84, 93)

ADD REPLYlink written 10 months ago by zachariasawaged0
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