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
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:
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.