[juan]

Split Read RNA-Seq mapping with bowtie?

How does Bowtie handle RNA-seq data, has anyone tried? Can it map split reads? Any plans to add split-read mapping functionality?

[ewilbanks]

Hi Lizzy,

I'd expect, oh, about 7-8 hours or so. Did it finish?

Thanks,
Ben

Hi Ben,

It did! Total run time was 9 hrs 22 min. Thanks!

Lizzy

[ewilbanks]

Hi Layla,

h_sapiens indexes the NCBI human reference contigs and h_sapiens_asm indexes the NCBI human reference assembly. Take a look at the scripts/make_h_sapiens.sh and scripts/make_h_sapiens_asm.sh files distributed with Bowtie to see exactly what fasta files were indexed and how.

People often prefer the assembly because the coordinates output by bowtie are more immediately useful (e.g., they correspond to the hg18 coordinates in the Genome Browser).

Thanks,
Ben

Hi Ben,

Thanks for this, I was confused about this as well. Might be a useful tidbit to put near the downloads on your website? Thanks for all the support. Bowtie rocks!

Lizzy

[Ben Langmead]

How does Bowtie handle RNA-seq data, has anyone tried? Can it map split reads? Any plans to add split-read mapping functionality?

Hi Juan,

Check out TopHat (linked to from the sidebar of the Bowtie website). TopHat was written by Cole Trapnell and it implements a layer on top of Bowtie that handles spliced alignments, along with several other aspects of alignment to the transcriptome and calling junctions.

Hope that helps.

Thanks,
Ben

[forrest]

Hi Ben,
Can Bowtie do DNA methylation aligment? I haven't found in your manual. How to define the parameter?

Thank you!

[para_seq]

Setting: Number of 'N's allowed in the reads

Hi, Ben,

I ran Bowtie for reads from Illumina sequencing. It is a very good pieces of software for its high speed and relatively small memory usage. I have two related questions. 1. I wonder if there is a switch in Bowtie to filter out reads that contain more than certain number of 'N' s. 2. How many 'N's in each read (e.g. 35-nt long) do people usually allow for Illumina data? Thank you.

[Ben Langmead]

Can Bowtie do DNA methylation aligment? I haven't found in your manual. How to define the parameter?

There are ad hoc ways of using Bowtie to do DNA methylation alignment, e.g., you can index and query the strands of your target genome separately and change the bases to mimic the bisulfite reaction (Cs -> Ts, for the most part). Refinements to this scheme are also possible (e.g. treat lone Cs and CpGs differently when mimicking the bisulfite reaction). As usual, the two main problems are loss of signal (more mismatches -> fewer reads align) and signal bias (depending on the conversion scheme, there might be an inherent coverage bias toward methylated or toward unmethylated sites).

I am also looking at trying to support bisulfite more directly in the future.

Thanks,
Ben

[SillyPoint]

Circular genomes

Does Bowtie make any provision for circular genomes?

I could fake it out by appending N-1 bases from the start of the reference onto the end of it, where N is the read length. But I'd need a separate index for each value of N.

Or perhaps not. If I use a large N, some reads will map both to the real position near the start, and to the same sequence in the appended copy. Running with --best and -k 1 (the default), I should get a single report at one of those positions.

But it would be a lot cleaner just to have a --circular option.

--TS

[Ben Langmead]

But it would be a lot cleaner just to have a --circular option.

You're correct that there is no --circular option. Your workaround is good, though.

Thanks,
Ben

[Ben Langmead]

1. I wonder if there is a switch in Bowtie to filter out reads that contain more than certain number of 'N' s.

In effect, that's what the -v/-n settings do. Ns count as mismatches, so if the alignment policy is "-v 2", reads with more than 2 Ns will not align. If Bowtie's options don't do exactly what you need, you could also write a very simple script that filters out reads according to your own criteria beforehand.

2. How many 'N's in each read (e.g. 35-nt long) do people usually allow for Illumina data? Thank you.

That depends very much on the quality of the data. Some sets of reads have many, many Ns, and some have Ns systematically at certain positions. My advice is to try various parameters and see what seems to give the best result.

Thanks,
Ben

[para_seq]

In effect, that's what the -v/-n settings do. Ns count as mismatches, so if the alignment policy is "-v 2", reads with more than 2 Ns will not align. If Bowtie's options don't do exactly what you need, you could also write a very simple script that filters out reads according to your own criteria beforehand.



That depends very much on the quality of the data. Some sets of reads have many, many Ns, and some have Ns systematically at certain positions. My advice is to try various parameters and see what seems to give the best result.

Thanks,
Ben

Thank very much, Ben.

[lix]

Hi Ben,

Thank you for Bowtie. It seems to be widely used and I am running it recently. There are some option settings I'm confused and I hope you can give me some kind suggestions:

I am mapping the ChIP-seq reads(raw sequences) to the reference genome(homo sapiens) and I want to the length of reads to be 25bp, with 2 mismatches to be allowed. So, I choose the option " --best -r -3". Is this option setting correct for mapping? If not, how the option should be setted?
I also build a reference genome index using "bowtie-build" option and is there any difference using this compared with the pre-built indexes on the website?

Thanks,

-lix

[Ben Langmead]

Hi lix,

I am mapping the ChIP-seq reads(raw sequences) to the reference genome(homo sapiens) and I want to the length of reads to be 25bp, with 2 mismatches to be allowed. So, I choose the option " --best -r -3". Is this option setting correct for mapping?

For raw reads, yes, use "-r". For allowing up to 2 mismatches, use "-v 2". It's also reasonable to use "--best"; it's noticeably slower than the default mode, but you might prefer having the best-ness guarantee.

I also build a reference genome index using "bowtie-build" option and is there any difference using this compared with the pre-built indexes on the website?

No difference. If you build an index using any of the scripts in the "scripts" subdirectory of the bowtie package, you should end up with an index that's effectively identical to the corresponding pre-built index. All pre-built indexes are built with bowtie-build.

Thanks,
Ben

[lix]

Ben, thanks for your so kind reply. I'm also wondering whether the "-3" option for the length of reads setting is correct. For example, if the length of my reads is 36bp and I want to the length just to be 25bp, so I set "-3 11" . Is that correct?

Thanks again,
lix

[Ben Langmead]

Ben, thanks for your so kind reply. I'm also wondering whether the "-3" option for the length of reads setting is correct. For example, if the length of my reads is 36bp and I want to the length just to be 25bp, so I set "-3 11" . Is that correct?

Yes, "-3 11" will turn an 36bp read into a 25bp read with 11 bases trimmed from the (3') end. To trim from the front (5') end, use "-5".

Thanks,
Ben