[maker-devel] advanced repeat masking library constructions & rna-seq assembly choices

[Salim Bougouffa]

Thank you all for your responses.

Regards,
/SB

On Mon, 8 May 2017, 18:50 Jiang, Ning, <jiangn at msu.edu> wrote:

> Hi Salim,
>
>
> I am sorry to learn about the issues. it depends on the quality of your
> genome assembly for how many intact LTR elements you would get; however, 16
> seems too low to me.
>
>
> The inner and LTR sequence file should NOT be empty. Some times the issue
> could be due to that the initial sequence name is long and complicated. If
> that's the case for your sequences, you might want to simplify your
> sequence name (only including letters and numbers) and try again.
>
>
> We are working on an automatic pipeline for LTR collection, if everything
> goes smoothly, it should be available in two to three months.
>
>
> Best wishes,
>
>
> Ning
> ------------------------------
> *From:* Campbell, Michael <mcampbel at cshl.edu>
> *Sent:* Sunday, May 7, 2017 9:24 PM
> *To:* Carson Holt
> *Cc:* Salim Bougouffa; maker-devel at yandell-lab.org List; Jiang, Ning
> *Subject:* Re: [maker-devel] advanced repeat masking library
> constructions & rna-seq assembly choices
>
> Hi SB,
>
> I’ve added Ning Jaing to this email. She has put great effort into
> updating this protocol recently and will be able to address your questions
> better than I can.
>
> Ning, would you mind helping out with this?
>
> Thanks,
> Mike
>
> On May 7, 2017, at 9:17 PM, Carson Holt <carsonhh at gmail.com<mailto:
> carsonhh at gmail.com>> wrote:
>
> Michael can you answer the second question (Michael wrote the protocol, so
> I CC’d him).
>
> With respect to the first question. Expression level is not necessarily
> relevant to the annotation process (so no MAKER does not look at read
> coverage). Instead we use the transcript assemblies to identify introns via
> splice aware alignment (yes it is the introns and not the exons we care
> about). Trinity has a nice option called jaccard_clip which avoids false
> merging of neighboring transcripts (mostly occurs in fungi where UTR can
> overlap). Merging of transcripts will cause extra introns to be assigned as
> hints as well as potential overextension of UTR during final polishing
> steps. The jaccard_clip option is the main reason we recommend Trinity. If
> Stringtie has a similar option, then it can be used as well.
>
> Thanks,
> Carson
>
>
>
> On May 4, 2017, at 12:37 AM, Salim Bougouffa <mjfi2sb3 at gmail.com<mailto:
> mjfi2sb3 at gmail.com>> wrote:
>
> Hi,
>
> I am attempting to annotate a plant genome. I have a couple of questions:
>
> 1) RNA-seq assembly
> a) I assembled my RNA-seq data using Trinity and StringTie. The two
> produce drastically different numbers. When I compare the two assemblies
> for each sample using TransRate, StringTie produces a higher score. for
> most of the assemblies. I see in all of the threads that you recommend
> Trinity but doesn't trinity produce way too many transcripts (even after
> chucking out the "bad" ones using transrate).
> b) During hint creation in MAKER, does it take into account that different
> transcripts have different read coverage (expression levels). I guess my
> question is should I filter transcripts that have a small read coverage.
>
> 2) Repeat Masking
> I am following the advanced repeat library construction tutorial (
> http://weatherby.genetics.utah.edu/MAKER/wiki/index.php/Repeat_Library_Construction-Advanced).
> The initial steps find 15 sequences for the LTR and 159 for MITE. But, when
> I get to the perl DIR_CRL/CRL_Step4.pl step, both output files
> (Inner_Seq_For_BLAST.fasta, lLTRs_Seq_For_BLAST.fasta) are empty.
>
> a) are these numbers normal because I was expecting a lot more than 16 for
> the LTR?
> b) I don't get any errors when I run CRL_Step4.pl yet no output. What's
> going on?!
>
> Many thanks,
> /SB
> --
>
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