Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Query regarding RNA-Seq hit/read count

    Hi everyone!

    I am running an RNA-Seq experiment and was wondering if anyone could help me with a particular query i had.

    So my RNA-Seq sample had 29,775,906 reads.

    I ran this sample through tophat (no coverage search, unstranded, w/bowtie2). I checked the logs folder in the output and got the following stats :-

    [2013-04-04 10:57:02] Preparing reads
    left reads: min. length=50, max. length=50, 29767088 kept reads (8818 discarded)

    Now, 29767088 + 8818 = 29,775,906 . So until now it makes sense.

    From the bowtie files i got the following information :-

    In,bowtie.left_kept_reads.log

    29767088 (100.00%) were unpaired; of these:
    4316521 (14.50%) aligned 0 times
    17118264 (57.51%) aligned exactly 1 time
    8332303 (27.99%) aligned >1 times

    In,bowtie.left_kept_reads_seg1.log
    4316521 (100.00%) were unpaired; of these:
    3198237 (74.09%) aligned 0 times
    1037571 (24.04%) aligned exactly 1 time
    80713 (1.87%) aligned >1 times

    In bowtie.left_kept_reads_seg2.log
    4316521 (100.00%) were unpaired; of these:
    3177510 (73.61%) aligned 0 times
    1069186 (24.77%) aligned exactly 1 time
    69825 (1.62%) aligned >1 times

    So at this point , correct me if i am wrong, we can conclude :-

    total reads aligned 1 time = 17118264 + 1069186 + 1037571 = 19225021
    total reads aligned >1 time = 8332303 + 80713 + 69825 = 8482841
    But then i converted the accepted_hits.bam file in a .sam file. On this .sam file I did the following two operations :-

    awk '{print $1}' 11129_nc_u.sam | sort | uniq -u | wc -l

    gave me 21638486 (i.e total number of uniques)

    and

    awk '{print $1}' 11129_nc_u.sam | sort | uniq -d | wc -l

    gave me 5519946 (i.e. total duplicates.)

    Now why is the number of uniques from the .sam folder (21638486 ) greater than 19225021 (i.e. obtained from the logs folder)? In the same breath, why is teh number of duplicates from .sam folder (5519946) less than the number in the logs folder (8482841) ?

    Can someone please clarify this point for me?

    Thanks in Advance

Latest Articles

Collapse

  • seqadmin
    Essential Discoveries and Tools in Epitranscriptomics
    by seqadmin


    The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...
    Yesterday, 07:01 AM
  • 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

ad_right_rmr

Collapse

News

Collapse

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