Can I use multiple reference sequences in my assembly?

Yes. The assembler can handle multiple reference sequences but they need to be combined into a sequence list document before assembly. Do this using Sequence→Group Sequences into a List... and then select this list as the reference sequence. Geneious will then try all reads against all references in a single operation.  

If you want to map all reads to each reference sequence in separate assemblies, use the Workflow "Map reads to each reference sequence".  This is available under Tools->Workflows. 

If you have a lot of shorter reference sequences, consider combining them using the concatenation tool into a single sequence with N spacers between each sequence.  This will prevent a situation you can get where you run out of open file handles and you’ll also just get a single contig document produced rather than many (See this article for more information)

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Comments

  • Avatar
    Tracy Campbell

    What if we have multiple sequence lists that we want aligned to a reference separately (i.e. >1000 sequence list documents) and have a sequence list of our reference sequences we want to map to?  I think the way it is set up currently, I would have to add my reference sequences to each of the NGS sequence read lists that I want assembled, but with that many sequences it would be arduous to add them all.  

  • Avatar
    Helen Shearman

    Yes, this is possible.  You do not need to add the reference sequence(s) to each of the lists of sequence reads.

    The reference sequences will need to be in a single sequence list.  Select the sequence list of reference sequences and one or more sequence lists of reads and go to Align/Assemble→Map to Reference.  In the dropdown box next to "Reference sequence" select the sequence list containing the reference sequences.  To align each of the sequences lists independently to the reference sequences, in the map to reference assembly options select "Assemble each sequence list separately".  Geneious will assemble each of the sequence lists to each of the reference sequences in the sequence list of reference sequences.

  • Avatar
    Masse T.

    Hi,
    I want to map my RADseq data against Wolbachia whole genome to remove it from my dataset, how should I run mapping in this case so that the original fastq.gz file will be trimmed (left without Wolbachia genome)?

  • Avatar
    Masse T.

    Any answers please?

  • Avatar
    Hilary Miller

    When you set up the mapping, check the option "Save unused reads" under the results settings. This will give you a list of all the reads that did not match the Wolbachia genome. If you want a separate list of all the reads that do match, check "Save used reads".

  • Avatar
    Masse T.

    Dear Hilary,
    Thanks for your reply, I did this and then I noticed that for those reads that were not mapped against the reference genome the unused read has longer sequence length than the original read! judging by the original length of the read and the reference genome, I supposed the unused read is the sum of both and not just the remains of my original read file! this is not what I want, I simply want to remove the mitochondrion+Wolbachia WG from each one of my 61 ddRADseq reads. Do you have any suggestion for this case please?

  • Avatar
    Hilary Miller

    I think you'll need to send us some screenshots showing your data and what has ended up in the unused reads file, so that we can understand what your datasets look like. Its probably best if you can open a support ticket with us via the Contact us button in Geneious.