3
9
Entering edit mode
7.9 years ago
bdeonovic ▴ 210

Does anyone have any recommendations for

What aligners to use for long reads (e.g. Pac Bio, Oxford nanopore)?

Currently I am using gmap, blast, lastz, and blasr (I'm going to try out blat as well).

What particular parameters to use for these aligners to map long reads well.

For pac bio long reads I have been using gmap. In several presentations on oxford nanopore I have heard that they have used either blast or lastz to map reads. These programs have tons of parameters to tweak. I'd like to hear what some good choices are .

RNA-Seq alignment • 14k views
12
Entering edit mode
7.9 years ago
lh3 33k

Nick Loman recommended LAST (not lastz) for mapping oxford nanopore reads. As I have tried, it worked very well at least for small genomes. A few others also seconded. For PacBio, its native mapper blasr is the proven tool, though some complain it is a bit slow. Another option is bwa-mem from github (self advertisement). It is better than the latest release 0.7.10. Bwa-sw is not as good. I reviewed a manuscript where the authors claim that older bwa-mem and bowtie2 don't work well (probably true) and that gem works well, but at my hand gem does not really work at all with their setting. I haven't heard from the authors yet. As to others, blat uses non-overlapping 11bp seeds (by default) and gmap use 20bp (or 24bp) exact seeds. Even if they work, you may need a lot of tuning. According to the email provided by coreruhno, STAR does not work with raw pacbio reads. It only works with CCS reads. Smalt, BBmap and MUMmer are all claimed to work with PacBio reads, but I have not tried myself. Finally, blast, lastz and blastz should also work provided that you use the right settings.

Probably I need to do a brief survey.

Self promotion: PacBio/Oxford Nanopore alignment with bwa-mem:

git clone https://github.com/lh3/bwa.git
cd bwa; make
bwa index ref.fa
bwa mem -x pacbio ref.fa pacbio.fq > aln.sam
bwa mem -x ont2d ref.fa ont-2D.fq > aln.sam

0
Entering edit mode

Excellent, I see an option for oxford nanopore 2D reads, what if I want to align template and complement reads?

1
Entering edit mode

You can still use the 2d option, but the sensitivity will be lower. On the one dataset I have tried, last is slightly (IMO) better than bwa-mem. It is worth trying last as well.

0
Entering edit mode

One more thing, when I tried the command you suggested (bwa mem -x ont2d ref.fa ont-2D.fq > aln.sam) I got something along the lines of index not found.

0
Entering edit mode

Hi Heng! Is the ont2d mode new? I haven't seen that yet. I have some BLAST results of the public ONP E. coli dataset; I'll have to give bwa-mem a try and compare. Thanks for the constant support of and updates to BWA!

0
Entering edit mode

But BWA would be useful only for DNA sequencing, right? For RNA-seq I guess STAR would work nicer.

1
Entering edit mode
7.9 years ago
Stoploss25 ▴ 10

I really like STAR as an aligner, and recommend it if you have a high-memory pc or cluster. It can be configured for use with long reads. See here.

0
Entering edit mode
7.9 years ago
Dgg32 ▴ 90

Just look this up yesterday, go take a look at bwa's long read Version: bwa-sw