Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • High gap penalty in BWA

    I'm using BWA MEM to align paired-end fastq files to two reference sequences (50bp and 80bp):

    >ref1
    TCGTAACGCAAGTTGGATACTCTCGA******************************GGATGTTGCCGTCCTCCTTGAAGT

    >ref2
    TCGTAACGCAAGTTGGATACTCTCGATTGCAAGTAGTCGATTGCATTGTCAATCTAGGATGTTGCCGTCCTCCTTGAAGT

    These two sequences are idential except the middle bit. I then used samtools to filter out properly paired reads from each sequence based on identifiers but surprisingly it showed overwhelming majority of reads have the number of matches that correspond to **ref2** and 0 properly paired reads for **ref1**

    I verified this result by repeating the process for each sequence individually and got the same output:

    ```
    $ samtools flagstat mysam.sam

    1055596 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 secondary
    0 + 0 supplementary
    0 + 0 duplicates
    20960 + 0 mapped (1.99% : N/A)
    1055596 + 0 paired in sequencing
    527798 + 0 read1
    527798 + 0 read2
    0 + 0 properly paired (0.00% : N/A)
    20934 + 0 with itself and mate mapped
    26 + 0 singletons (0.00% : N/A)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)
    ```
    I then tried setting high values for gap opens and extension penalties in order to limit the chance that hits from alignments of **ref1** reads result in origin of **ref2**. By doing this, I now can see some properly paired reads from **ref1**. However, one thing I don't understand is that the total number of reads goes up while this number should go down because of gap penalties.

    Here are the result (I combined ref1 and ref2 in one file called refx.fasta):

    ```
    bwa mem -O 20 -E 20 refx.fasta R1.fastq.gz R2.fastq.gz > mysam.sam

    samtools flagstat mysam.sam

    1117216 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 secondary
    61620 + 0 supplementary
    0 + 0 duplicates
    1116527 + 0 mapped (99.94% : N/A)
    1055596 + 0 paired in sequencing
    527798 + 0 read1
    527798 + 0 read2
    1053928 + 0 properly paired (99.84% : N/A)
    1054666 + 0 with itself and mate mapped
    241 + 0 singletons (0.02% : N/A)
    6 + 0 with mate mapped to a different chr
    5 + 0 with mate mapped to a different chr (mapQ>=5)```

    ```
    bwa mem -O 30 -E 30 refx.fasta R1.fastq.gz R2.fastq.gz > mysam.sam

    samtools flagstat mysam.sam

    1121698 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 secondary
    66102 + 0 supplementary
    0 + 0 duplicates
    1121007 + 0 mapped (99.94% : N/A)
    1055596 + 0 paired in sequencing
    527798 + 0 read1
    527798 + 0 read2
    1053912 + 0 properly paired (99.84% : N/A)
    1054662 + 0 with itself and mate mapped
    243 + 0 singletons (0.02% : N/A)
    6 + 0 with mate mapped to a different chr
    5 + 0 with mate mapped to a different chr (mapQ>=5)
    ```

    Can someone explain to me:

    1. Is it normal to see 0 properly paired reads in this case?
    2. Why does disallowing reads with open and extension gaps increase the total of reads?

    Many thanks
    Last edited by annguyen; 01-15-2021, 02:23 AM.

Latest Articles

Collapse

  • 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
  • seqadmin
    Techniques and Challenges in Conservation Genomics
    by seqadmin



    The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

    Avian Conservation
    Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
    03-08-2024, 10:41 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, Yesterday, 06:37 PM
0 responses
11 views
0 likes
Last Post seqadmin  
Started by seqadmin, Yesterday, 06:07 PM
0 responses
10 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-22-2024, 10:03 AM
0 responses
51 views
0 likes
Last Post seqadmin  
Started by seqadmin, 03-21-2024, 07:32 AM
0 responses
68 views
0 likes
Last Post seqadmin  
Working...
X