Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • HISAT2 vs. TopHat2: Discrepancies between Hg19 & Hg38

    Hello everyone,

    I have around 150 RNA-Seq datasets created using the Lexogen SENSE mRNA-Seq Library Prep Kit for Illumina, as well as around 50 Trueseq Illumina samples. I aligned one sample using both HISAT2 and TopHat2 for both Hg19 and Hg38. The reason for that is that I wish to run all samples with Hg38 (since it is the newest reference) and HISAT2, but using Hg19 I get a far better alignment rate:

    Lexogen Sample:
    HISAT2 (Hg19): Paired Rate = 82.68%, Overall Rate = 90.31%
    HISAT2 (Hg38): Paired Rate = 73.87%, Overall Rate = 81.01%
    TopHat2 (Hg19): Paired Rate = 74.74%, Overall Rate = 87.4%
    TopHat2 (Hg38): Paired Rate = 77.68%, Overall Rate = 87.7%

    It is interesting to notice, that TopHat2 does not seem to be negatively affected by the change of reference. On the contrary it actually "likes" it.
    This got even more strange, when I run a control sample created with the TrueSeq Illumina Kit and got the following results:

    Trueseq Sample:
    HISAT2 (Hg19): Paired Rate = 94.86%, Overall Rate = 97.22%
    HISAT2 (Hg38): Paired Rate = 93.15%, Overall Rate = 95.47%
    TopHat2 (Hg19): Paired Rate = 93.46%, Overall Rate = 96.50%
    TopHat2 (Hg38): Paired Rate = 88.07%, Overall Rate = 97.00%

    I can accept a difference in the alignment rate as "random" if its less than 5% but a drop from 90.31% to 81.01% I cannot accept. Has anyone tested HISAT2 on those different reference genomes and if so had similar results? I have been struggling with this for a long time so any help is greatly appreciated!!

    Additional Info:
    - The whole analysis was run on the Galaxy Platform
    - The Lexogen Samples are strand-specific (second strand) and the Trueseq samples are unstranded
    - I tested 4 additional samples (2 Lexogen + 2 Trueseq) gaining similar results
    - The references were downloaded from the USCS directly

    Thanks in advance!
    Sbamo

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
18 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 10:19 PM
0 responses
22 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 09:21 AM
0 responses
17 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-04-2024, 09:00 AM
0 responses
49 views
0 likes
Last Post seqadmin  
Working...
X