[ensembl-dev] whole chromosome alignment with LastZ

[Alice Iob]

Dear Matthieu,

Thank you very much for your swift reply.

With post-processing do you mean generate a consensus alignment from sam files (if I split the query, align it and then set the output in sam)? or something else?

Also, I was wondering if you are planning to release a whole genome alignment of Triticum durum and T. dicoccoides in the future.

Thank you for your help.

Alice Iob
PhD student
Plant and Animal Genomics Program
CRAG, Centre for Research in Agricultural Genomics
Campus UAB - CRAG Building | 08193 Cerdanyola | BARCELONA
Office: 3.01
Tel. +34 935636600 ext 3351
________________________________
Da: Matthieu Muffato [muffato at ebi.ac.uk]
Inviato: giovedì 26 marzo 2020 10.10
A: Ensembl developers list; Alice Iob
Oggetto: Re: [ensembl-dev] whole chromosome alignment with LastZ


Dear Alice,

I've seen that you have sent this question to the lastz-users list and Bob replied with some tips.

We don't get this error we get because we have engineered a workflow around LastZ that does what Bob suggests, i.e. the masking, the splitting with overlap and its post-processing, and some chaining.

It's not easily usable by external people, though, because it heavily relies on an infrastructure that's built in Ensembl. But Bob gave some options to run those steps by yourself, which should address the issue.

Best,
Matthieu

On 25/03/2020 11:36, Alice Iob wrote:
Good morning,

I am a Phd student woking on plant genomics and I am stuggling with an issue regarding LastZ.
I choose to use LastZ because it was used for plant genome alignments in Ensembl Plants, so I hope that someone who succesfully used it before can help me with this.

I am trying to align two references genomes from very close species: I have two FASTA files,
representing the same chromosome in the two species, each around 800Mb long, with at least one long repetitive region.

the command I am using:

lastz target.fasta query.fasta --notransition --step=20 --maxwordcount=70 ‑‑exact=20 --chain --gapped --ambiguous=iupac --rdotplot=plot --format=differences > alignment.differences

I always get the same error:

FAILURE: in add_segment()
table size (4,869,542,152 for 101,448,794 segments) exceeds allocation limit of 4,294,967,279;
consider raising scoring threshold (--hspthresh or --exact) or breaking your target sequence into smaller pieces.

I tried several strategies to overcome this issue:
increasing values of exact up to 100
using values of hspthresh up to 10 000
adding --seed=match12
dividing my target sequence in two (one multiFASTA file)
working with just half chromosome (400Mb)
set the parameters as they were set to align T. aestivum and A. tauschii (https://plants.ensembl.org/mlss.html?mlss=9814)

Still, I get the same error.

Just a few times I was able to get an output (e.g. when exact=100), but it is always more than 700Gb big, thus, even if the file is generated, I run out of memory and I can not work on it.

I also used LastZ_32 but the process gets killed without giving me any info.

I was wondering if you can help me with this issue, maybe I am not using properly some of the options, or give me some advice on how to proberly deal with this alignment.

Thank you.

Alice Iob
PhD student
Plant and Animal Genomics Program
CRAG, Centre for Research in Agricultural Genomics
Campus UAB - CRAG Building | 08193 Cerdanyola | BARCELONA
Office: 3.01
Tel. +34 935636600 ext 3351



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--
Matthieu Muffato, Ph.D.
Ensembl Compara Principal Developer
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
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