The k-mers in the ends of the reads could be short adapter remnants not identified by the adapter clipper. Typically they get soft-clipped by the aligner, but you should get rid of them if you use BBDuk in trimbyoverlap mode.
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Originally posted by pmiguel View PostMammals have teeny little exons spread out over 10's-100's of kilobases of the the genome. Mapping RNA (which has the introns spliced out) reads to the genome isn't a good way to determine insert size. And only getting 50% of the reads to map "concordantly" doesn't seem so bad. How is bowtie2 going to handle reads spanning a splice site?
If you want to determine your insert sizes, try aligning your reads to a long (spliced) transcript instead of genomic DNA. In my experience with the MiSeq and HiSeq, your sizes will look like all the very shortest library products were sequenced preferentially.
--
Phillip
Comment
-
I think the problem lies in the bowtie2 command:
-1 R1.paired.fq -2 R1.unpaired.fq
-1 R1.paired.fq -2 R2.paired.fq
Comment
-
Originally posted by Brian Bushnell View PostI think the problem lies in the bowtie2 command:
Based on the filenames, it appears that you are telling it to treat read1's as pairs with each other, using the output of something like Trimmomatic that produced 4 output files. You should be doing something like this:
Although, I'm not really sure why ANY of the pairs were concordant.
Comment
-
Originally posted by dpryan View PostGood catch! I share your wonderment that any were concordant. I'm also surprised it didn't throw an error that the files were of different lengths.
Anyway now I almost have my inner size and I am trying to align them by TopHat. I will update this thread by the result of my TopHat aligning.
Thanks all!
Comment
Latest Articles
Collapse
-
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...-
Channel: Articles
04-22-2024, 07:01 AM -
-
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...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 11:49 AM
|
0 responses
15 views
0 likes
|
Last Post
by seqadmin
Yesterday, 11:49 AM
|
||
Started by seqadmin, 04-24-2024, 08:47 AM
|
0 responses
16 views
0 likes
|
Last Post
by seqadmin
04-24-2024, 08:47 AM
|
||
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
61 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
60 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
Comment