We are going to divide the group in 4 groups.
Samples to assemble:
Sample_104B
Sample_105B
Sample_119B
Sample_120A
Trimming: Enabled
Sample_104B
Sample_105B
Sample_119B
Sample_120A
Trimming: Dissabled
DF19
DF5
Sample_103A
Sample_104B
Sample_105B
Sample_118A
Sample_119B
Sample_120A
spike_Z32_rep1
spike_Z32_rep2
Trimming: Enabled
DF19
DF5
Sample_103A
Sample_104B
Sample_105B
Sample_118A
Sample_119B
Sample_120A
spike_Z32_rep1
spike_Z32_rep2
Trimming: Dissabled
The inputs are in:
/var/scratch/Practical4_Example/reads
Trinity requires
#!/bin/bash -e
#SBATCH -p batch
#SBATCH -w taurus
#SBATCH -o log/list_files.%N.%j.out
#SBATCH -n 8
#SBATCH --mem=35G
module load trinity/v2.6.6
left="sample1.r1.fastq.gz,sample2.r1.fastq.gz,sample3.r1.fastq.gz"
right="sample1.r2.fastq.gz,sample2.r2.fastq.gz,sample3.r2.fastq.gz"
Trinity --trimmomatic --seqType fq --max_memory 35G --left $left --right $right --CPU 8 --output my_trinity_output --full_cleanup
echo "DONE"using the full_cleanup to remove at the end all the temporary files for trinity. The parameter CPU is consistent with -n in #SBATCH. Likewise, the memory should be consistent.
- What are the steps that trinity is doing? What do you think each step does?
- Can you try the same argumetns in trimmomatic as used on the previous excercises?
Get the size of all the transcripts and get a distribution of the sizes. Compare that with the reference that we had before (selected_refseq1.0.fasta).
Get the N50. You need to sort the transcript lengths and the cummulative values.
To compare this assemblies, we are going to map them using gmap:
module load gmap/2015-12-31
If you have problmes running the version in the cluster, try the version in: /home/rgonzalez/gmap/bin
Copy the transformed_coordinates.fasta to your folder and create an index for gmap
gmap_build -d transformed_coordinates -D gmap transformed_coordinates.fasta
Align your assembly to the Trinity assembly.
gmap --min-intronlength=20 --format=gff3_gene --npaths=1 --ordered --min-identity=0.95 -d transformed_coordinates -D ../References/gmap trinity-mini_test.Trinity.fasta > trinity-mini_test.Trinity.gff
GMAP version 2018-05-30 called with args: gmap.avx2 --min-intronlength=20 --format=gff3_gene --npaths=1 --ordered --min-identity=0.95 -d transformed_coordinates -D ../References/gmap trinity-mini_test.Trinity.fasta
We need to sort the GFF file in coordinates, a tool that can be used for this is genometools http://genometools.org/tools/gt_gff3.html /home/rgonzalez/gt/bin/
gt gff3 -tidy yes -retainids yes -sort yes trinity-mini_test.Trinity.gff > trinity-mini_test.sorted.Trinity.gff
We can do this with all the transcriptomes that where generated.
You can visualize all the transcriptome assemblies at the same time with igv. Look for the differences between the original references (transformed_coordinates.gff) and your assembly. Also compare them with the assemblies of other groups.
Genome tools can be used to produce a plot of a particular region, for example: chr1B:320,232-345,985
gt sketch -format pdf -seqid chr1B -start 320232 -end 345985 output1.pdf trinity-mini_test.sorted.Trinity.gff
That particular region looks like this:
