Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • roll
    replied
    Originally posted by dpryan View Post
    Assuming that you're using the mm10 reference, you can download the repeatmasker output here (mm9 is here). The general idea is to extract the type of feature(s) you want from the repeatmasker .out file and convert that to bed format and use "bedtools intersect ..." to get a count of how many reads align there. There are many other ways to do this, but that should work.

    In fact, a more straight-forward way might be simply to run cufflinks on your alignments and then intersect the novel transcripts it finds with the repeatmasker output file. That might end up being easier.
    from the repeat list, i am trying to use the ones on forward strand. do you know how to extract this from the .out file?
    The column headers are like
    SW perc perc perc query position in query matching repeat position in repeat
    score div. del. ins. sequence begin end (left) repeat class/family begin end (left) ID

    687 17.4 0.0 0.0 chr1 3000002 3000156 (194195276) C L1_Mur2 LINE/L1 (4310) 1567 1413 1
    917 21.4 11.4 4.5 chr1 3000238 3000733 (194194699) C L1_Mur2 LINE/L1 (4488) 1389 913 1
    215 3.1 0.0 3.0 chr1 3000734 3000766 (194194666) + (TTTG)n Simple_repeat 2 33 (0) 2

    Leave a comment:


  • dpryan
    replied
    Originally posted by roll View Post
    I did the check.
    As you suggested the order is different.

    The only differences are that
    i have chrM in fa file and MN in the gtf file.
    Also in the gtf file i have lots of 'NT_123456' type of entries which i do not what it is.

    Do these differences cause any problem?
    That will generally still cause issues with tophat. If you downloaded your bowtie indices from iGenomes (likely via a link on the bowtie webpage), then they came with an appropriate reference annotation file. Just use that one.

    Leave a comment:


  • roll
    replied
    I did the check.
    As you suggested the order is different.

    The only differences are that
    i have chrM in fa file and MN in the gtf file.
    Also in the gtf file i have lots of 'NT_123456' type of entries which i do not what it is.

    Do these differences cause any problem?

    Leave a comment:


  • dpryan
    replied
    No need to manually do that

    Just:
    Code:
    grep ">" reference.fa
    on the reference fasta file to get a list of the contigs and then:
    Code:
     cat annotation.gtf | cut -f 1 | sort | uniq
    on the GTF or GFF file. They should be the same, possibly with a different order (and the output from the grep command will all start with ">", which you can ignore).

    Leave a comment:


  • roll
    replied
    Do you mean manually checking if all chromosomes are there? i do not know what contigs are or how to check them.

    I think chrX and Y are missing in the gtf file. But how can I find information for missing chromosomes and complete it?

    Leave a comment:


  • dpryan
    replied
    That should be sufficient, just make sure that the annotation doesn't mention any chromosomes/contigs missing from the reference fasta file (I can't recall if that's the case or not).

    Leave a comment:


  • roll
    replied
    I am using mm9. And I downloaded the bowtie index from their webpage. I had to change the chr names as ensembl uses the 1 instead of chr1. But i don't know whether just adding chr in front of the chromosomes names will sort it out. Should I do other changes in addition?

    Leave a comment:


  • dpryan
    replied
    If you're aligning against mm10, then don't use an annotation file from Ensembl (the chromosome names are different). That will cause no end of issue If you use the Ensembl annotation, just align against the genome that you can download from Ensembl (the Ensembl annotation is better anyway).

    Yeah, that was probably me, there's a large overlap between the people here and on biostars.

    Leave a comment:


  • roll
    replied
    Originally posted by dpryan View Post
    Just download the GTF annotation and use gtf2bed from bedops. That should keep the gene names (or some other useful identifier).

    Edit: The GTF annotation is available from the UCSC table browser, in case you weren't aware
    Thanks Devon,
    Very helpful so far.
    I downloaded this annotation from ensembl. From biomart i simply left the filters sections empty and used the whole output as my list. This is right, no?

    PS. i just asked a question in biostar about flagstat and it was answered by you i think but i am not sure.

    Leave a comment:


  • dpryan
    replied
    Just download the GTF annotation and use gtf2bed from bedops. That should keep the gene names (or some other useful identifier).

    Edit: The GTF annotation is available from the UCSC table browser, in case you weren't aware

    Leave a comment:


  • roll
    replied
    Originally posted by dpryan View Post
    Assuming that you're using the mm10 reference, you can download the repeatmasker output here (mm9 is here). The general idea is to extract the type of feature(s) you want from the repeatmasker .out file and convert that to bed format and use "bedtools intersect ..." to get a count of how many reads align there. There are many other ways to do this, but that should work.

    In fact, a more straight-forward way might be simply to run cufflinks on your alignments and then intersect the novel transcripts it finds with the repeatmasker output file. That might end up being easier.
    Thanks a lot. This has been very helpful so far. i am trying what you have suggested and will let you know how it goes.

    Do you know where i can download a genes and coordinates in a bed format? (Alternatively how can i assign gene names to my bed file)?

    Leave a comment:


  • lh3
    replied
    BTW, you can find more detailed repeatMask results here:

    http://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/rmsk.{sql,txt.gz}

    It is easy to convert this file to BED, I believe.

    Leave a comment:


  • dpryan
    replied
    Assuming that you're using the mm10 reference, you can download the repeatmasker output here (mm9 is here). The general idea is to extract the type of feature(s) you want from the repeatmasker .out file and convert that to bed format and use "bedtools intersect ..." to get a count of how many reads align there. There are many other ways to do this, but that should work.

    In fact, a more straight-forward way might be simply to run cufflinks on your alignments and then intersect the novel transcripts it finds with the repeatmasker output file. That might end up being easier.

    Leave a comment:


  • roll
    replied
    Originally posted by roll View Post
    great, so we are getting there

    I would like to have something with numbers rather than examining it as visual.

    how do I get the repeatmasker output from uscs? do i upload my sam file and then tick repeatmask option?

    What about bedtools? which option i should look for?
    Originally posted by dpryan View Post
    Ah, actually repeat masking the reads won't do what you want then. What you want to do is align your reads to the mouse genome and then download the repeat masker output from UCSC. There are then a number of ways to compare your alignments to where the repeats are (e.g., just visually inspecting things with IGV or using bedtools or something similar to intersect the alignments).
    I have used tophat2 for mapping. Shall i use the bam file? Other outputs are

    accepted_hits.bam
    align_summary.txt
    deletions.bed
    insertions.bed
    junctions.bed
    logs
    prep_reads.info
    unmapped.bam

    Leave a comment:


  • roll
    replied
    Originally posted by roll View Post
    great, so we are getting there

    I would like to have something with numbers rather than examining it as visual.

    how do I get the repeatmasker output from uscs? do i upload my sam file and then tick repeatmask option?

    What about bedtools? which option i should look for?
    I have used tophat2 for mapping. Shall i use the bam file? Other outputs are

    accepted_hits.bam
    align_summary.txt
    deletions.bed
    insertions.bed
    junctions.bed
    logs
    prep_reads.info
    unmapped.bam

    Leave a comment:

Latest Articles

Collapse

  • seqadmin
    Multiomics Techniques Advancing Disease Research
    by seqadmin


    New and advanced multiomics tools and technologies have opened new avenues of research and markedly enhanced various disciplines such as disease research and precision medicine1. The practice of merging diverse data from various ‘omes increasingly provides a more holistic understanding of biological systems. As Maddison Masaeli, Co-Founder and CEO at Deepcell, aptly noted, “You can't explain biology in its complex form with one modality.”

    A major leap in the field has
    ...
    02-08-2024, 06:33 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 02-21-2024, 08:52 AM
0 responses
32 views
0 likes
Last Post seqadmin  
Started by seqadmin, 02-20-2024, 08:57 AM
0 responses
23 views
0 likes
Last Post seqadmin  
Started by seqadmin, 02-14-2024, 09:19 AM
0 responses
55 views
0 likes
Last Post seqadmin  
Started by seqadmin, 02-12-2024, 03:37 PM
0 responses
462 views
0 likes
Last Post seqadmin  
Working...
X