How to begin genome-guided Trinity

[Farbod Emami]

Hi Dear Trinity experts,

Sorry for this very simple question,

I have used my 3 males and 3 females (12 paired-end fastq files) non-model fish for de-novo assembly using Trinity.

Now, I want to run Genome-guided approach but I do not know how to began to create "read alignments to Trinity as a coordinate-sorted bam file" and  "coordinate sorted bam file"

I have find a related species genome from here and I want to use STAR for my other steps.

Do I must align each samples (e.g: first sample1-left.fastq & sample1-right.fastq and then sample2 . . . ) separately to the genome ? 

Do I must use "bowtie-build" command first on the Genome file ?

[Tiago Hori]

I would not use the genome of closely-related species for this. It may add more confusion than benefits, unless that are super closely related.

 

T.

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[Farbod Emami]

Dear Dr Tiago , Hi.

So what kind of genome I must use for genome guided ?

I intend to check the DEGs in this situation.

On Tuesday, August 23, 2016 at 9:23:57 PM UTC+4:30, Tiago Hori wrote:

I would not use the genome of closely-related species for this. It may add more confusion than benefits, unless that are super closely related.

 

T.

 

From: trinityrn...@googlegroups.com [mailto:trinityrn...@googlegroups.com] On Behalf Of Farbod Emami
Sent: August 23, 2016 1:45 PM
To: trinityrnaseq-users <trinityrn...@googlegroups.com>
Subject: [trinityrnaseq-users] How to begin genome-guided Trinity

 

Hi Dear Trinity experts,

 

Sorry for this very simple question,

 

I have used my 3 males and 3 females (12 paired-end fastq files) non-model fish for de-novo assembly using Trinity.

 

Now, I want to run Genome-guided approach but I do not know how to began to create "read alignments to Trinity as a coordinate-sorted bam file" and  "coordinate sorted bam file"

 

I have find a related species genome from here and I want to use STAR for my other steps.

 

Do I must align each samples (e.g: first sample1-left.fastq & sample1-right.fastq and then sample2 . . . ) separately to the genome ? 

 

Do I must use "bowtie-build" command first on the Genome file ?

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[Farbod Emami]

Please have a look at this:

https://www.researchgate.net/figure/262788782_fig6_Figure-1-Assembly-of-the-guppy-reference-transcriptome-A-Flowchart-describing-read

[Tiago Hori]

It is not clear from the figure, but I will assume that when they say draft genome, they mean the Guppy draft genome. That’s fine. It does not have to be a final genome. However, using a closely related species is another story. For one, you assuming most genes are organized in the same way in the genome (unlikely), 2 you are assuming the gene duplications is equal between both genomes (unlikely), you are assuming genes are conserved enough for the genome to actually help you (possible, but unlikely).

 

Why not just do a de-novo assembly? That’s one of Trinity’s main strengths.

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[Farbod Emami]

Very nice answer, as usual. Thank you

As I have some transcripts in my DEGs that has no blast hits n blasting against NCBI nr database, i think that using a genome guided wit gar fish or zebrafish and search for the genomic position of them may be show me something new.

but according to your guidance, this approach is use-less

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[Mark Chapman]

Hi Farbod,
As you already have a de novo assembly just blast the DEGs against the genome and hopefully they will hit an annotated gene. Although if there was no hit in the first place I think it's unlikely you'll get a lot from searching the genome.
Best, Mark

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[Farbod Emami]

Dear Dr. Mark, Hi.

I know how to locally blast my DEGs against NCBI nr or nt or other databases, 

But I do not know how to "blast the DEGs against the genome" ? 

I have a fasta file that contains my DEGs sequences and for example I have the Gar fish genome I have provided from its link in the beginning of the question.

Did I must make this reference genome as a blastable database, first?

thank you.

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[Mark Chapman]

Yes that would be my suggestion if the genome isn't in ncbi.

Dear Dr. Mark, Hi.

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[Farbod Emami]

Thank you,

and what if " the genome is exist in ncbi" ?

and I think the gar genome is in the NCBI (here), am I right or I am missing something ?

Dear Dr. Mark, Hi.

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[Mark Chapman]

if its already in ncbi then your blast will include it as a reference, you dont need to make your own blastable db.

Thank you,

Dear Dr. Mark, Hi.

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[Mark Chapman]

use this:

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=OGP__7918__72169

to blast directly against the gar genome

[Farbod Emami]

Dear Mark and Tiago,

I know that using such genome guided is useless but just for curiosity and learning the mapping procedures from you, I have used STAR program 6 times (I have two treatments, 3 biological replication for each, so 6 left and right pairs fastq), and now I have 6 "*Aligned.sortedByCoord.out.bam" files.

1- How can I use these 6 files in the genome guided script provided via Trinity (here)? Did I must merge them? or runing the related Trinity command six times separately? 

2- Can I use these .bam files to check some quality or aligning representation?

This is one of my scripts for align RNA-Seq Reads to the genome with STAR:

STAR   --genomeDir ./   --runThreadN 24   --readFilesIn    '/home/F1-left.fastq'     '/home2/F1-right.fastq'  --outFileNamePrefix  F1 --outSAMtype   BAM SortedByCoordinate

Dear Dr. Mark, Hi.

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[Farbod Emami]

Many thanks !

Thank you,

Dear Dr. Mark, Hi.

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[EDUtainment TV]

Hi all,

I ran the genome guided trinity by using following command and completed until following steps and is still running. But the succeeded percentage get stacked in same position from last 2 days. My question is, these kind of things happen in this trinity assembly process or what, also if i get an error can i restart the trinity run from the last completed step, if yes then how can i. Could anyone please help me on this. Thank you.

[prabinb@biolab ~]$ Trinity --genome_guided_bam /data/tmp/StarElmap/ElsantaAligned.sortedByCoord.out.bam --genome_guided_max_intron 100000 --max_memory 50G --CPU 12


--------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------------


-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NW_004443499.1/0/258_1156.trinity.reads
-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NW_004443501.1/0/2_695.trinity.reads
-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NW_004443501.1/0/870_1016.trinity.reads
-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NW_004443502.1/0/68_1040.trinity.reads
-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NW_004443503.1/0/2_1011.trinity.reads
-writing to Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff/NC_015206.1/0/1_155688.trinity.reads
CMD: touch Dir_ElsantaAligned.sortedByCoord.out.bam.minC1.gff.ok
##
Done
##

Saturday, August 27, 2016: 00:10:10    CMD: touch partitions.ok
Saturday, August 27, 2016: 00:10:10    CMD: find Dir_* -name '*reads' > read_files.list
Saturday, August 27, 2016: 00:10:10    CMD: touch read_files.list.ok
Saturday, August 27, 2016: 00:10:10    CMD: /opt/trinityrnaseq/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file read_files.list --CPU 1 --max_memory 1G  --seqType fa --trinity_complete --full_cleanup  > trinity_GG.cmds
Saturday, August 27, 2016: 00:10:10    CMD: touch trinity_GG.cmds.ok
Saturday, August 27, 2016: 00:10:10    CMD: touch trinity_GG.cmds.ok


--------------------------------------------------------------------------------
------------ Trinity Phase 2: Assembling Clusters of Reads ---------------------
--------------------------------------------------------------------------------

Saturday, August 27, 2016: 00:10:10    CMD: /opt/trinityrnaseq/trinity-plugins/parafly/bin/ParaFly -c trinity_GG.cmds -CPU 12 -v
Number of Commands: 2703
succeeded(2696)   99.741% completed.