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Question: Which reference to map against for RNA-Seq using HISAT2
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gravatar for skhan
13 months ago by
skhan • 40
skhan • 40 wrote:

Hi all,

I am trying to determine differential expression between two test conditions - in rat. The first step would be to map the sequencing data to a rat reference of some sort, and I would like to use HISAT2 (locally installed on my machine) along with the Ensemble rat genome.

Which reference file(s) do I map against?

1) Do I use the hisat2 index for rn6 as is: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/rn6.tar.gz ?

Or

2) Do I need to generate my own hisat2 index from the Ensemble reference genome (ftp://ftp.ensembl.org/pub/release-86/fasta/rattus_norvegicus/dna/Rattus_norvegicus.Rnor_6.0.dna_sm.toplevel.fa.gz) and annotations file (ftp://ftp.ensembl.org/pub/release-86/gtf/rattus_norvegicus/Rattus_norvegicus.Rnor_6.0.86.gtf.gz)

Thanks, skhan
rna-seq hisat2 • 1.0k views
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modified 13 months ago by Jennifer Hillman Jackson ♦ 25k • written 13 months ago by skhan • 40
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gravatar for Jennifer Hillman Jackson
13 months ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hi,

The rn6 reference genome is sourced from UCSC so has different chromosome identifier formats than the Ensembl version, so it would require a different reference annotation file. UCSC has an Ensemble track based on the rn6 build. Check the date on it to see if it is current enough for your needs - it is probably older than the one you linked to.

Using the Ensembl genome/annotation versions are also an option.

For either, install the genome and create the indexes with the Data Managers (fetch genome, sam, picard, hisat2). These are in the tool shed. See the DM tutorial here for how-to: https://galaxyproject.org/admin/#data-resources

The Chrom identifier mismatch FAQ here explains why it is important to make sure that data is all from the same build when doing an analysis (plus how to check/adjust for mismatches, when possible): https://galaxyproject.org/support/chrom-identifiers/

Hope that helps! Jen, Galaxy team
ADD COMMENT • link written 13 months ago by Jennifer Hillman Jackson ♦ 25k
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