Question: align_and_estimate_abundance after assembly in Trinity
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gravatar for nasreenbano91
6 weeks ago by
nasreenbano910 wrote:

i have transcriptome data (R1 and R2 of Cestrum diurnum & R1 AND R2 of Cestrum nocternum). I have to perform de novo assembly through Trinity to find out the gene differences in between these 2 species. i did quality filter, assembly and align_and_estimate_abundance through trinity. till assembly everything was f9 bt after align_and estimate result we get the files of isomers and genes. when we open this file i get the 99% FPKM, expected_count and TPM value is zero and rest 1% having extremely high values which is not possible. again we perform the same process bt again we get the same values of FPKM expected_count and TPM like this:

gene_id transcript_id(s)        length  effective_length        expected_count  TPM     FPKM
TRINITY_DN10000_c0_g1   TRINITY_DN10000_c0_g1_i1        309.00  45.06   0.00    0.00    0.00
TRINITY_DN10001_c0_g1   TRINITY_DN10001_c0_g1_i1        264.00  24.19   0.00    0.00    0.00
TRINITY_DN10001_c0_g2   TRINITY_DN10001_c0_g2_i1        480.00  159.73  0.00    0.00    0.00
TRINITY_DN10002_c0_g1   TRINITY_DN10002_c0_g1_i1        422.00  115.82  0.00    0.00    0.00

when i open the error file(.e) , get the warning like below given which i don't understand-

tput: No value for $TERM and no -T specified
CMD: /app/setups/trinityrnaseq-2.2.0/util/support_scripts/get_Trinity_gene_to_trans_map.pl /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta > /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.gene_trans_map
CMD: touch /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.bowtie.started
CMD: bowtie-build /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.bowtie
CMD: touch /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.RSEM.rsem.prepped.started
CMD: rsem-prepare-reference  --transcript-to-gene-map /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.gene_trans_map /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fas
ta.RSEM
CMD: set -o pipefail && bowtie -q --all --best --strata -m 300 --chunkmbs 512 -X 800 -S -p 4 /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.bowtie -1 /scratch/sbag/nasreen/vinayak_new/cd1_1.fq -2 /scratch/sbag/nasreen/cd1_2.fq
| samtools view -F 4 -S -b -o RSEM_CD1.bowtie.bam -
# reads processed: 8323031
# reads with at least one reported alignment: 1031 (0.01%)
# reads that failed to align: 8322000 (99.99%)
Reported 1405 paired-end alignments to 1 output stream(s)
CMD: touch RSEM_CD1.bowtie.bam.ok
CMD: rsem-calculate-expression  --paired-end   -p 4    --no-bam-output --bam RSEM_CD1.bowtie.bam /scratch/sbag/nasreen/vinayak_new/Trinity1/Trinity.fasta.RSEM RSEM_CD1
Warning: Detected a read pair whose two mates have different names--ST-E00192:475:H53GLCCXY:2:1101:20192:7532 and ST-E00192:475:H53GLCCXY:2:1101:15503:6970!
Warning: Detected a read pair whose two mates have different names--ST-E00192:475:H53GLCCXY:2:1101:30401:12033 and ST-E00192:475:H53GLCCXY:2:1101:29843:11839!
Warning: Detected a read pair whose two mates have different names--ST-E00192:475:H53GLCCXY:2:1101:24657:13685 and ST-E00192:475:H53GLCCXY:2:1101:18639:13562!
Warning: Detected a read pair whose two mates have different names--ST-E00192:475:H53GLCCXY:2:1101:23997:17219 and ST-E00192:475:H53GLCCXY:2:1101:24150:17237!

what should i have to do? please help

ADD COMMENTlink modified 20 days ago • written 6 weeks ago by nasreenbano910

I added markup to your post for increased readability. You can do this by selecting the text and clicking the 101010 button. When you compose or edit a post that button is in your toolbar, see image below:

101010 Button

In addition, I modified your post from a 'tutorial' to a 'question' and edited the tags to include "trinity" which is quite crucial in your question.

ADD REPLYlink written 6 weeks ago by WouterDeCoster23k
0
gravatar for nasreenbano91
4 weeks ago by
nasreenbano910 wrote:

find the solution.

ADD COMMENTlink written 4 weeks ago by nasreenbano910

If you did, then describe it here in detail, instead posting this cryptic line. It would be useful for others who later find this thread by search.

ADD REPLYlink written 4 weeks ago by genomax37k
0
gravatar for nasreenbano91
20 days ago by
nasreenbano910 wrote:

The output file of quality filter was not equal that's why this problem was happening. After making R1 and R2 reads equal through merging and splitting script this problem can be easily resolved.

ADD COMMENTlink written 20 days ago by nasreenbano910
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