• What will be the minimum number of biological replicates (plant samples) and how many biological replicates are needed for RNA-Seq using HIseq ?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
One more question about the "replicate" definition; if you have a cDNA from a biological sample (for a RNA-seq experiment) and you use it into two PCR reactions with different barcodes (like the one you do before the Illumina run), the barcode1 and barcode2 samples from the same cDNA can be considered as "replicates".
Suppose you want statistical power in your data, and you want to test differential gene expression in programs like DESeq (where it wants replicates to check the so called "shot-noise"); in this case, can they be considered good "replicates"?
Thanks
Comment
-
Originally posted by cascoamarillo View PostOne more question about the "replicate" definition; if you have a cDNA from a biological sample (for a RNA-seq experiment) and you use it into two PCR reactions with different barcodes (like the one you do before the Illumina run), the barcode1 and barcode2 samples from the same cDNA can be considered as "replicates".
Suppose you want statistical power in your data, and you want to test differential gene expression in programs like DESeq (where it wants replicates to check the so called "shot-noise"); in this case, can they be considered good "replicates"?
Thanks
Comment
-
Yes, these replicates are only testing the reproducibility of the sequencing protocol, which is extremely high. Biological reproducibility is what we're interested in. I would just pool the data from technical replicates as a single biological replicate.
Comment
-
I would do no less than three independent biological replicates (i.e. RNA isolated from separate biological samples) of each condition. You can get meaningful results from 2 biological reps, but you are really restricting yourself by doing this, particularly on a HiSeq where you can easily multiplex and still get a lot of reads per sample even on a single lane.
Comment
Latest Articles
Collapse
-
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 -
-
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...-
Channel: Articles
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
22 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
24 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
19 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
52 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment