Tophat with Bowtie2 long index

Question

[BDK_compbio]

I am getting some error while running tophat2. I used bowtie2 for building indices using the following command

bowtie2-build --large-index <refrence> <output directory>

It generates indices with suffix bt21 (instead of bt2). Bue when I am running tophat it is giving me following error

Could not find Bowtie 2 index files (.*.bt2)

[Devon Ryan]

Which version of tophat2? I think only the most recent version supports large indexes (it doesn't actually say in the release notes).

[BDK_compbio]

I am using tophat/2.0.12 which is the latest version I guess.

[Devon Ryan]

Hello sbdk82!

It appears that your post has been cross-posted to another site: SEQanswers

This is typically not recommended as it runs the risk of annoying people in both communities.

[BDK_compbio]

Thanks for the suggestion. I posted on both sites thinking that I would get quick answer.

[Manvendra Singh]

I ran it like

/usr/local/bin/bowtie2-build -f ~/Genome_fasta/hg19refseq.fa ~/homo_bowtie2_index/hg19

where hg19 is base index prefix and hg19refseq.fa is reference sequence

I got

hg19.1.bt2
hg19.3.bt2
hg19.rev.1.bt2
hg19.2.bt2
hg19.4.bt2
hg19.rev.2.bt2

in the given directory

And TopHat2 always runs very well using them.

[BDK_compbio]

I also ran like that, but it gave me the following error

Error: Reference sequence has more than 2^32-1 characters!  Please divide the reference into batches or chunks of about 3.6 billion characters or less each and index each independently.

That's why I tried --large-index option. I am now using BWA-MEM and STAR instead of bowtie+tophat

[Manvendra Singh]

May be, you try with TopHat 2.0.8 version which works for me.

[BDK_compbio]

I am using tophat 2.0.12

[BDK_compbio]

Actually the reference file is too large, so bowtie-index won't work if you don't use --large-index option. The current version of tophat does not support the large index. Did you use --large-index parameter? Maybe the size of your reference file is not large enough.

[Manvendra Singh]

which reference genome you are using?

I did with Human, Chimp, Gorilla, Rhesus, cynomolgus, mouse etc, trference genome and never face any problem

[Devon Ryan]

None of those have a large enough genome to require large-genome support, which is good since tophat can't yet do that.

[bioinfo_student]

I am using a prebuild index (.bt2 files) of hg19 for bowtie2, but every runs has an error that says it is too large

Out of memory allocating the ebwt[] array for the Bowtie index. Please try again on a computer with more memory.

I know hg19 is too large, and even the fastQ file I am using is also large. But is not there anyway out to run bowtie 2 on this?

[Devon Ryan]

Firstly, please start a new thread for things like this rather than posting questions as a comment.

Secondly, you can certainly use bowtie2 for hg19, you just need to use a computer with more RAM (as the error message said).

[bioinfo_student]

Oops sorry for not starting a new thread. I am new here and somewhere it said, for discussions use the comment tab!

OK so I need more RAM. How much is least RAM required to run a mapping for hg19? My PC has 8GB RAM

I am quite new to this, so please consider this as a genuine doubt! And yes I will make sure I post doubts as new threads from next one!

THANKS!

[Devon Ryan]

I'm a little surprised that that's not enough, though I suspect you have other things running. I would expect that 12 gigs would suffice.

[bioinfo_student]

Thank you for the reply! Even I was wondering why? I tried closing all other programs and running it again! No luck!