Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Isolating Longest Sequences from FASTA file - rmdup, dedeupe, VSEARCH help

    Greetings, [this bonked on submission, sorry if repost, can't see previous]

    I have a large list of fasta sequences (not paired end) from which I want to isolate the longest version of each "sequence" (obviously the longest one has a different sequence). For example:

    input.fa:
    >Seq_1
    AAACCCGGGTTT

    Seq_2
    CCCGGG

    output_keep.fa:
    >Seq_1
    AAACCCGGGTTT

    output_ditch.fa:
    >Seq_2
    CCCGGG

    I tried a number of approaches using a mock dataset containing mismatches, exact copies, and known truncations from either end of the long form - "test_dups.fa":

    (1) dedupe.sh in SAMtools
    dedupe.sh test_dups.fa test_dups_dedupe.fa outd=test_dups_removed.fa

    this didn't work: its output didn't make sense, and an exact duplicate pair was removed completely (didn't leave behind one of them, which is bad news).

    (2) with the ac=f option
    dedupe.sh test_dups.fa test_dups_dedupe.fa outd=test_dups_removed.fa ac=f

    that only removed the exact copy pair, again not leaving one copy in file.

    (3) clustering by overlap
    dedupe.sh test_dups.fa test_dups_dedupe.fa outd=test_dups_removed.fa ac=f mo=50 c pc csf=stats.txt outbest=test_best.fa mcs=1

    didn't work (retained were not longest examples).

    Then I moved on to VSEARCH, to sort by size, then form clusters and output the "seed":

    (4) vsearch clustering
    --cluster_fast dups.fa --centroids test_unique_1.0.fa --id 1.0
    oddly, this kept a truncation of the longest sequence, but was close...

    (5) using the iddef = 0, to ignore end gaps
    vsearch --cluster_fast dups.fa --centroids test_unique_1.0.fa --id 1.0 --iddef 0

    this did not ignore the end gaps penalties as expected.

    (6) Align two seqs and see what the perceived idenity is
    vsearch --allpairs_global dups_9_10.fa --acceptall --alnout test_aln.fa --iddef 0

    the resulting alignment shows 100% identity, as expected with the iddef 0 option.

    Why are those sequences not in the same cluster then?

    Searching for help on "remove duplicates" is a disaster here, so I hope I can get some help. One consideration is to merge the sequences with 100% identity for the "overlap", but those tools merge from one end of the read, as is used to combine paired end reads. The other consideration is spending a day writing a script. I reasoned I can not be the first person to do this.

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
31 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 10:19 PM
0 responses
33 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 09:21 AM
0 responses
28 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-04-2024, 09:00 AM
0 responses
53 views
0 likes
Last Post seqadmin  
Working...
X