Trinity De Novo assembly with PacBio Iso-seq correction
Mayson Lin
I have obtained Illumina paired-end short reads and PacBio ccs Iso-seq. I attempted to do De Novo asssembly with iso-seq long reads correction.
This is my script
"Normalization complete. See outputs:
Mayson Lin
Brian Haas
the long reads are given to Trinity separately from the short reads.
If you're running the genome guided Trinity, then the long reads are provided aligned in a separate bam file via parameter: --long_reads_bam, and the aligned illumina reads are provided via: --genome_guided_bam
On Sat, Sep 11, 2021 at 9:45 PM Mayson Lin <sli...@fiu.edu> wrote:
>
> Or the question is, how do I know if the --long_reads works ? The transcriptome result is N50:1015 nt , BUSCO:96.1%, which is not ideal quality. Also, does "--long_reads" can be applied to genome-guided mode? I tried, it can be completed, but I am not sure if it really took long reads input at all.
>
> On Saturday, September 11, 2021 at 9:26:07 PM UTC-4 Mayson Lin wrote:
>>
>> Greetings,
>> I have obtained Illumina paired-end short reads and PacBio ccs Iso-seq. I attempted to do De Novo asssembly with iso-seq long reads correction.
>> This is my script
>>
>> "Normalization complete. See outputs:
>> /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/insilico_read_normalization/Blowfly-male-Phormia_R1_001.fastq.gz.PwU.qtrim.fq_ext_all_reads.normalized_K25_maxC200_minC1_maxCV10000.fq
>> /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/insilico_read_normalization/Blowfly-female-Phormia_R2_001.fastq.gz.PwU.qtrim.fq_ext_all_reads.normalized_K25_maxC200_minC1_maxCV10000.fq
>> Friday, September 10, 2021: 22:09:44 CMD: touch /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/insilico_read_normalization/normalization.ok
>> Converting input files. (in parallel)Friday, September 10, 2021: 22:09:44 CMD: cat /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/insilico_read_normalization/left.norm.fq | seqtk-trinity seq -r -A -R 1 - >> left.fa
>> Friday, September 10, 2021: 22:09:44 CMD: cat /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/insilico_read_normalization/right.norm.fq | seqtk-trinity seq -A -R 2 - >> right.fa
>> Friday, September 10, 2021: 22:10:31 CMD: touch right.fa.ok
>> Friday, September 10, 2021: 22:10:36 CMD: touch left.fa.ok
>> Friday, September 10, 2021: 22:10:37 CMD: touch left.fa.ok right.fa.ok
>> Friday, September 10, 2021: 22:10:37 CMD: cat left.fa right.fa > /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa
>> Friday, September 10, 2021: 22:10:54 CMD: touch /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa.ok
>> Friday, September 10, 2021: 22:11:03 CMD: cp /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa.wLR
>> Friday, September 10, 2021: 22:11:20 CMD: cat /home/data/FLAG/Phormia_transcriptome/longread.fasta | sed 's/>/>LR\$\|/' >> /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa.wLR
>> Friday, September 10, 2021: 22:11:21 CMD: touch /home/data/FLAG/Phormia_transcriptome/trinity_out_dir/both.fa.wLR.ok"
>
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--
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Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas
Mayson Lin
I took your feedback and used genome-guided mode with long reads, but this is what I get wrong, it appears long reads is too short
Trinity version: Trinity-v2.13.2
-ERROR: couldn't run the network check to confirm latest Trinity software version.
Sunday, September 12, 2021: 16:58:58 CMD: /usr/local/bin/util/support_scripts/ensure_coord_sorted_sam.pl /scratch/mdegenna/slin023/stringtie/ShortAlignedSorted.out.bam
-appears to be a coordinate sorted bam file. ok.
Sunday, September 12, 2021: 16:58:58 CMD: java -Xmx64m -XX:ParallelGCThreads=2 -jar /usr/local/bin/util/support_scripts/ExitTester.jar 0
Sunday, September 12, 2021: 16:58:58 CMD: java -Xmx4g -XX:ParallelGCThreads=2 -jar /usr/local/bin/util/support_scripts/ExitTester.jar 1
----------------------------------------------------------------------------------
-------------- Trinity Phase 1: Clustering of RNA-Seq Reads ---------------------
----------------------------------------------------------------------------------
-found paired-end aligned read. Running in paired-end mode.
* [Sun Sep 12 16:58:58 2021] Running CMD: java -jar /usr/local/src/picard.jar AddOrReplaceReadGroups I=/scratch/mdegenna/slin023/stringtie/LongSorted.out.bam O=/scratch/mdegenna/slin023/star/trinity_out_dir/LongSorted.out.bam.RGLR.bam RGID=PBLR RGLB=lib2 RGPL=pacbio RGPU=unit2 RGSM=pacbio VALIDATION_STRINGENCY=LENIENT
INFO 2021-09-12 20:58:59 AddOrReplaceReadGroups
********** NOTE: Picard's command line syntax is changing.
**********
********** For more information, please see:
**********
********** The command line looks like this in the new syntax:
**********
********** AddOrReplaceReadGroups -I /scratch/mdegenna/slin023/stringtie/LongSorted.out.bam -O /scratch/mdegenna/slin023/star/trinity_out_dir/LongSorted.out.bam.RGLR.bam -RGID PBLR -RGLB lib2 -RGPL pacbio -RGPU unit2 -RGSM pacbio -VALIDATION_STRINGENCY LENIENT
**********
20:59:00.232 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/usr/local/src/picard.jar!/com/intel/gkl/native/libgkl_compression.so
[Sun Sep 12 20:59:00 UTC 2021] AddOrReplaceReadGroups INPUT=/scratch/mdegenna/slin023/stringtie/LongSorted.out.bam OUTPUT=/scratch/mdegenna/slin023/star/trinity_out_dir/LongSorted.out.bam.RGLR.bam RGID=PBLR RGLB=lib2 RGPL=pacbio RGPU=unit2 RGSM=pacbio VALIDATION_STRINGENCY=LENIENT VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false
[Sun Sep 12 20:59:00 UTC 2021] Executing as sli...@n099.panther.net on Linux 3.10.0-1127.el7.x86_64 amd64; OpenJDK 64-Bit Server VM 11.0.10+9-Ubuntu-0ubuntu1.18.04; Deflater: Intel; Inflater: Intel; Provider GCS is not available; Picard version: 2.25.7
INFO 2021-09-12 20:59:00 AddOrReplaceReadGroups Created read-group ID=PBLR PL=pacbio LB=lib2 SM=pacbio
[Sun Sep 12 20:59:03 UTC 2021] picard.sam.AddOrReplaceReadGroups done. Elapsed time: 0.06 minutes.
Runtime.totalMemory()=2155872256
-- Skipping CMD: samtools merge /scratch/mdegenna/slin023/star/trinity_out_dir/merged_wRGLR.bam /scratch/mdegenna/slin023/star/trinity_out_dir/ShortAlignedSorted.out.bam.norm_200.bam /scratch/mdegenna/slin023/star/trinity_out_dir/LongSorted.out.bam.RGLR.bam && samtools index /scratch/mdegenna/slin023/star/trinity_out_dir/merged_wRGLR.bam, checkpoint [/scratch/mdegenna/slin023/star/trinity_out_dir/merged_wRGLR.bam.ok] exists.
Sunday, September 12, 2021: 16:59:03 CMD: find Dir_* -name '*reads' > read_files.list
Sunday, September 12, 2021: 16:59:10 CMD: /usr/local/bin/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file read_files.list --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup --long_reads_mode > trinity_GG.cmds
Error, reads file listing: read_files.list is empty. This tends to happen when there were too few reads to assemble. at /usr/local/bin/util/support_scripts/write_partitioned_trinity_cmds.pl line 51.
Error, cmd: /usr/local/bin/util/support_scripts/write_partitioned_trinity_cmds.pl --reads_list_file read_files.list --CPU 1 --max_memory 1G --run_as_paired --seqType fa --trinity_complete --full_cleanup --long_reads_mode > trinity_GG.cmds died with ret 65280 at /usr/local/bin/Trinity line 2863.
main::process_cmd("/usr/local/bin/util/support_scripts/write_partitioned_trinity"...) called at /usr/local/bin/Trinity line 3672
main::write_trinity_partitioned_cmds("read_files.list", "trinity_GG.cmds", "GENOME_GUIDED_MODE") called at /usr/local/bin/Trinity line 3544
main::run_genome_guided_Trinity("/scratch/mdegenna/slin023/stringtie/ShortAlignedSorted.out.bam", "/scratch/mdegenna/slin023/stringtie/LongSorted.out.bam") called at /usr/local/bin/Trinity line 1411
I used minimap 2 to align PacBio ccs and sorted with samtools.
Here is my script of Trinity:
#!/bin/bashIB_40C_512G
#SBATCH --qos pq_mdegenna
#SBATCH --account iacc_mdegenna
# Node locationks
#SBATCH -p IB_40C_512G
# Number of nodeslog
#SBATCH -N 1
# Number of taskslarity-3.5.3
#SBATCH -n 18rinity-2.10.0
#SBATCH --output=log
singularity exec -e -B /scratch/mdegenna/slin023/star /home/slin023/image/trinitmodule load singularity-3.5.3 --genome_guided_bam /scratch/mdegenna/slin023/strimodule load trinity-2.10.0ut.bam --long_reads_bam /scratch/mdegenna/slin023/stringtie/LongSorted.out.bam --genome_guided_max_intron 10000 --max_memory 18G --CPUsingularity exec -e -B /scratch/mdegenna/slin023/star /home/slin023/image/trinityrnaseq.v2.13.2.simg Trinity --genome_guided_bam /scratch/mdegenna/slin023/stringtie/ShortAlignedSorted.out.bam --long_reads_bam /scratch/mdegenna/slin023/stringtie/LongSorted.out.bam --genome_guided_max_intron 10000 --max_memory 18G --CPU 18
Is there anyway to improve it?Brian Haas
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