Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • bvk
    replied
    Originally posted by GenoMax View Post
    Technically cuffdiff command as outlined above will work. But Devon has already warned you about the consequence in post #5.
    I used this in command line:

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    Now I want to run this from a python script. So, I gave a call in this way.

    do.call([cfg.tool_cmd("cuffdiff"), "-p", str(cfg.project["analysis"]["threads"]), "-b", str(cfg.project["genome"]["fasta"]), "-u", cfg.project["samples"][0]["files"]["merging_gtf"], "-L", str(cfg.project["phenotype"][0]), str(cfg.project["phenotype"][1]), "-o", output_folder] + [cfg.project["samples"][0]["files"]["bam"] cfg.project["samples"][1]["files"]["bam"]], cfg.project["analysis"]["log_file"])

    but I get an error: invalid syntax. Can you please help in this?

    Leave a comment:


  • bvk
    replied
    Originally posted by GenoMax View Post
    Technically cuffdiff command as outlined above will work. But Devon has already warned you about the consequence in post #5.
    Yes, I agree but I would like to know how to check the expression levels of each sample if I run this with merged bam file.

    Leave a comment:


  • bvk
    replied
    Originally posted by dpryan View Post
    You only have 2 samples anyway. Any sort of metric you'd get from each of the subfiles isn't terribly meaningful unless you're interested in looking at technical variance.
    Ok will see. thanks !! But I would like to know like how to check the expression levels of each sample if I run this with merged bam file

    Leave a comment:


  • GenoMax
    replied
    Technically cuffdiff command as outlined above will work. But Devon has already warned you about the consequence in post #5.

    Leave a comment:


  • bvk
    replied
    Originally posted by GenoMax View Post
    Arrange the labels (larval/early) according to their correspondence with the BAM files.

    Code:
    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam
    As you said If I run the following command

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    Now, If I want to find the expression levels for each sample I guess it is not possible, cz of the merged bam file.

    Is it possible to give labels as larval,early and giving bam files of 6 samples. Will this work?

    for eg:

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L embryo,larva -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

    Leave a comment:


  • dpryan
    replied
    You only have 2 samples anyway. Any sort of metric you'd get from each of the subfiles isn't terribly meaningful unless you're interested in looking at technical variance.

    Leave a comment:


  • bvk
    replied
    Originally posted by dpryan View Post
    No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.
    As you said If I run the following command

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    Now, If I want to find the expression levels for each sample I guess it is not possible, cz of the merged bam file.

    Is it possible to give labels as larval,early and giving bam files of 6 samples. Will this work?

    for eg:

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L embryo,larva -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

    Leave a comment:


  • bvk
    replied
    Originally posted by dpryan View Post
    No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.
    Thankyou !! I got it.

    Leave a comment:


  • bvk
    replied
    Originally posted by GenoMax View Post
    Arrange the labels (larval/early) according to their correspondence with the BAM files.

    Code:
    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam
    Yes, I understood. Thank you very much !!

    Leave a comment:


  • GenoMax
    replied
    Originally posted by bvk View Post
    so you say that it should look like this:

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    SRR493359_60_61_thout which has merged bam file of 59,60 and 61
    SRR493363_64_65_thout which has merged bam file of 63,64 and 65
    Arrange the labels (larval/early) according to their correspondence with the BAM files.

    Code:
    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    Leave a comment:


  • bvk
    replied
    Originally posted by dpryan View Post
    Aside from your syntax problems, you have a more serious issue in that you're not specifying the actual experiment correctly. SRR493359, SRR493360 and SRR493361 are from the same sample and should just be merged together into a single BAM file. Similarly SRR493363, SRR493364 and SRR493365 are from the same sample. So, you actually have a 1 vs. 1 sample comparison. Do NOT lump each of the files for each sample into a group, since then you're making fake replicates and will have largely meaningless results (of course, you're doing a 1 vs. 1 comparison, so the results aren't exactly robust to begin with).
    so you say that it should look like this:

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

    SRR493359_60_61_thout which has merged bam file of 59,60 and 61
    SRR493363_64_65_thout which has merged bam file of 63,64 and 65

    Leave a comment:


  • dpryan
    replied
    Originally posted by bvk View Post
    ok. now the below code looks fine I guess

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam
    No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.

    Leave a comment:


  • bvk
    replied
    Originally posted by GenoMax View Post
    The -L option you have used in your command is not two group names for three samples in each group.

    ", ../tophat/la/SRR493365_thout/accepted_hits.bam " It also looks like there is a space between , and the .. but that may be an illusion in the way the browser is displaying it.
    ok. now the below code looks fine I guess

    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

    Leave a comment:


  • dpryan
    replied
    Aside from your syntax problems, you have a more serious issue in that you're not specifying the actual experiment correctly. SRR493359, SRR493360 and SRR493361 are from the same sample and should just be merged together into a single BAM file. Similarly SRR493363, SRR493364 and SRR493365 are from the same sample. So, you actually have a 1 vs. 1 sample comparison. Do NOT lump each of the files for each sample into a group, since then you're making fake replicates and will have largely meaningless results (of course, you're doing a 1 vs. 1 comparison, so the results aren't exactly robust to begin with).

    Leave a comment:


  • GenoMax
    replied
    The -L option you have used in your command is not two group names for three samples in each group.

    ", ../tophat/la/SRR493365_thout/accepted_hits.bam " It also looks like there is a space between , and the .. but that may be an illusion in the way the browser is displaying it.

    Leave a comment:

Latest Articles

Collapse

  • seqadmin
    Current Approaches to Protein Sequencing
    by seqadmin


    Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
    04-04-2024, 04:25 PM
  • seqadmin
    Strategies for Sequencing Challenging Samples
    by seqadmin


    Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
    03-22-2024, 06:39 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 04-11-2024, 12:08 PM
0 responses
12 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 10:19 PM
0 responses
17 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 09:21 AM
0 responses
14 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-04-2024, 09:00 AM
0 responses
43 views
0 likes
Last Post seqadmin  
Working...
X