Closed iwangtoknow closed 6 years ago
Hi, when did you download the image? Which image are you using? I made some changes and maybe these can make problem.
Cheers Luigi
Hi, Luigi
lfaino/lorean <none> 153ab8ea611e 6 weeks ago 32.9GB
I can only provide you this, I just pull the last iprscan_rpMask, so the former image tag change to <none>
Can LoReAn auto-remove the folder route now? Thx WANG
hi, can you remove the image and run it again? i made a new image and you can check if the problem solvers.
Cheers luigi
Hi Luigi, A new error occurred.
PASA configuration file existed already: /data/LoReAn_20180716/run/PASA/alignAssembly.config --- skipping
###LOADING GFF3 FILE INTO DATABASE###
###UPDATING GFF3 FILE###
###PARSING OUTPUT###
Traceback (most recent call last):
File "/opt/LoReAn/code/lorean.py", line 550, in <module>
main()
File "/opt/LoReAn/code/lorean.py", line 397, in main
final_keep = grs.genename_last(final_output, args.prefix_gene, args.verbose, pasa_dir, dict_ref_name, "pasa")
File "/opt/LoReAn/code/getRightStrand.py", line 173, in genename_last
db1 = gffutils.create_db(out.name, ':memory:', merge_strategy='create_unique', keep_order=True, transform=transform_name)
File "/usr/local/lib/python3.5/dist-packages/gffutils/create.py", line 1288, in create_db
c.create()
File "/usr/local/lib/python3.5/dist-packages/gffutils/create.py", line 504, in create
self._populate_from_lines(self.iterator)
File "/usr/local/lib/python3.5/dist-packages/gffutils/create.py", line 569, in _populate_from_lines
for i, f in enumerate(lines):
File "/usr/local/lib/python3.5/dist-packages/gffutils/iterators.py", line 103, in __iter__
i = self.transform(i)
File "/opt/LoReAn/code/getRightStrand.py", line 231, in transform_name
fatureattgene = prefix_name + '_' + chrold + '_G_' + str(gene_count)
TypeError: unsupported operand type(s) for +: 'NoneType' and 'str'
Do you think Which one could be None
?
Thx WANG
Hi Wang, I think that i find the error. in 3 hours a new images should be ready to download and you can test it. let me know
Cheers Luigi
Hi Luigi,
I add a -n
parameter and the None value disappear anyway.
I finished a run but with I move assembly and RNA data to working dictionary.
So, maybe the problem fixed, I'm not sure.
LoReAn worked in my Fungi fine, Thanks for your excellent work.
Maybe next you can add more functional annotate step such as eggnog etc. That could be remote.
And some comparative analysis.
WANG
I get a few bad genes, I'm trying manually to fix them. I'll try the new image and reply to you.
No UTR and 5' should be extend.
No UTR and 5' should be extend.
Bad Gene
Not sure
Not sure
Not sure
Seems no problem also in bad list??
Maybe mRNA9837 is pseudo mRNA
HI Wang, can you send me the command that you use to run lorean? can you tell me which chemistry are you using to sequence cDNA?
Cheers Luigi
lorean.py \
-pr /data/ProteinEvidence/Asp_PDB_Uniprot6090.fasta \
-sr left.fq.right.fq \
-lr all_long_reads.fq \
-sp aspergillus_nidulans \
-mg -iprs -f -k -t 10 -q 3000 -n An \
BJCHM5.fa
left.fq
and right.fq
are my short reads transcripts data sequenced by Illumina Nova Seq PE-150, I have 3 sample and I cat
them together to left.fq
and right.fq
;
all_long_reaads.fq
is my long reads transcripts data sequenced by PacBio RSII I combine 2 cell data, the first cell only have a little num reads due to sequence sample, and the second cell produced more.
All long reads are *subreads.fq
, now I use IsoSeq3
polished my reads.
My genome also sequenced by RSII 2 cell data. And my short reads transcript data >60 million reads have a >95% unique mapping freq against my genome assembly.
The long sub_reads have many gap and SNP that is normal. THANKS Luigi
ps. I'm still download the latest iprscan_rpMask
image, I failed 5 times due to web connection error.
WANG
Hi Wang, i would suggest to use the option "-d" with your experiment using the image that you are downloading. It will automatically try to find sequencing adapter and strand the reads.
For your experiment, i would suggest to use CCS reads. I had very good results with CCS data.
Let me know Luigi
Thanks Luigi,
I use link to IsoSeq3!
the pipeline
Should I use polished reads or your recommendation of CCS data?
Luigi,
Sorry I don't understand the chemistry last reply. Both of my transcriptome sequencing experiments are general eukaryotic library, NOT the strand specific eukaryotic transcriptome library.
Can I also add the -d
option?
Hi Wang, yes you can use it. I thought that your sequencing was done by Nanopore and not PacBio for this I asked about chemistry. Can you see poly AAA in your sequencing output? The command would be:
lorean.py \ -d -pr /data/ProteinEvidence/Asp_PDB_Uniprot6090.fasta \ -sr left.fq.right.fq \ -lr all_long_reads.fq \ -sp aspergillus_nidulans \ -mg -iprs -f -k -t 10 -q 3000 -n An \ BJCHM5.fa
Hi Wang, few words on BAD GENES list. This are genes that do not start with M. I called them BAD GENES but they can be good genes.
Cheers Luigi
Thanks Luigi, Yes I can see primer+TTTTTTTTTTTT almost every sequence. The sequencing library is ployA enriched. You helped me a lot indeed. WANG
Hi Luigi, The dictionary error still there.
###RUNNING iASSEMBLER 18:11:40 25-07 ###
### STARTING ADAPTER ALIGNMENT AND READS ORIENTATION ###
100%|#################################| 224792/224792 [01:02<00:00, 3572.21it/s]
Traceback (most recent call last):
File "/opt/LoReAn/code/lorean.py", line 550, in <module>
main()
File "/opt/LoReAn/code/lorean.py", line 431, in main
args.max_intron_length, args.verbose, args.stranded)
File "/opt/LoReAn/code/manipulateSeq.py", line 171, in filterLongReads
filter_count = align.adapter_alignment(out_filename, adapter_aaa, scoring, align_score_value, out_filename_oriented, threads, min_length)
File "/opt/LoReAn/code/align.py", line 94, in adapter_alignment
with open(out_filename, "w") as output_handle:
FileNotFoundError: [Errno 2] No such file or directory: '/data/LoReAn_20180723/run//gmap_output/Data/long_reads/all_longreads.fq.longreads.filtered.oriented.fasta'
WANG
Hi Wang, can you check is the file /data/LoReAn_20180723/run//gmap_output/Data/long_reads/all_longreads.fq.longreads.filtered.oriented.fasta exists and is not empty?
Cheers Luigi
Hi Luigi,
I checked the file, the file all_longreads.fq.longreads.filtered.oriented.fasta
is NOT exists, and the dictionary Data
is also NOT exists.
I provide the long_reads data with -lr Data/long_reads/all_longreads.fq
, and I think you could cut the dictionary, output data to /data/LoReAn_20180723/run//gmap_output/all_longreads.fq.longreads.filtered.oriented.fasta
.
After the Error occurred, I made a symbolic link from Data/long_reads/all_longreads.fq
to ./all_longreads
. lorean.py -lr all_longreads.fq
is alright.
WANG
Hi Luigi,
I finished a second run. As you suggestion I add a -d
and provide CCS long reads data. A lot addition genes.
LOREAN GFF3 STATS -d and ccs last run ------------ | ------------- | ------------- parsed genome node DAGs | 10169 | 10378 sequence regions | 21 (total length: 31038331) | 22 (total length: 31036420) genes | 10147 | 10355 protein-coding genes | 10146 | 10355 mRNAs | 11453 | 10695 protein-coding mRNAs | 11452 | 10694 exons | 38142 | 33609 CDSs | 36273 | 32932 introns | 80067 | 68742
WANG
Are you satisfied with the results? Did you check the alignment of the long reads on the genome?
Cheers Luigi
Hi Luigi, I run the docker and meet this error:
I checked the code and last normal run dictionary, and I realized that I provided the file PacBio subreads.fastq with route
/Data/all_long_reads.fq
causing this problem.Another question should I provide polished long reads fastq file? I use PacBio Isoseq. Thx.