Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • just 2.4% by trimming at 27. i do not know if it's a good average for a 2x300bp, MiSeq 16s and a metaprofilig of another marker

    Comment


    • Is the command you posted, when you got the error the first time copy/paste from your terminal? In that case you simply made the mistake to only load the reverse reads
      That may also fit to the error message you got as you would have lost all mates. Interesting, however, that bbduk didn't complain and even produced two fastqs as output?!

      Originally posted by cnicolas View Post
      /data/umb/cichocki/bbmap/bbduk.sh in=/data/umb/cichocki/project2/bbduckdu11avril/project2_R2.fastq in=/data/umb/cichocki/project2/bbduckdu11avril/project2_R2.fastq out1=clean11avril.fastq out2=clean211avril.fastq qtrim=rl trimq=30

      Comment


      • Originally posted by WhatsOEver View Post
        Is the command you posted, when you got the error the first time copy/paste from your terminal? In that case you simply made the mistake to only load the reverse reads
        That may also fit to the error message you got as you would have lost all mates. Interesting, however, that bbduk didn't complain and even produced two fastqs as output?!
        Ah! Good eye...

        OK, so here's what's happening:

        Code:
        in=x.fq in=x.fq
        In1 is set as x.fq. Then, in1 is set as x.fq again (you can do this as many times as you want; BBTools all just overwrite the previous setting with the latest setting). Then, since 2 output files are specified, BBDuk assumes that the input file is interleaved and forces interleaved mode to true. That's a feature, by the way! But, I guess one that could potentially cause problems.

        Comment


        • --Hi,

          i have a big difference between results using bbduk.sh and trimmomatic trimming single-end reads, i have used the commands below, trimmomatic kept 99.78% survival reads whereas bbduk 91.76%. I don't know which to consider good or not.
          Which parameters do you use to use to trim in a good way single-reads ?

          thank you --

          java -Xmx10g -jar trimmomatic-0.36.jar SE -threads 8 -phred33 D3_464_S2_L001_R1_001.fastq.gz Out_D3_464_S2_L001_R1_001.fastq.gz ILLUMINACLIP:TruSeq3-SE.fa:2:40:15:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

          TrimmomaticSE: Started with arguments:
          -threads 8 -phred33 D3_464_S2_L001_R1_001.fastq.gz Out_D3_464_S2_L001_R1_001.fastq.gz ILLUMINACLIP:/home/jtazi/save/Trimmomatic-0.36/adapters/TruSeq3-SE.fa:
          2:40:15:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
          Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
          Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
          ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
          Input Reads: 49512700 Surviving: 49401731 (99.78%) Dropped: 110969 (0.22%)

          bbduk.sh Xmx8g in=D3_464_S2_L001_R1_001.fastq.gz out=D3_464_S2_L001_R1_001_trimmed.fastq.gz ref=resources/adapters.fa threads=8 k=13 ktrim=r useshortkmers=t mink=5 qtrim=rl minlength=36 trimq=27

          BBDuk version 36.11
          Set threads to 8
          maskMiddle was disabled because useShortKmers=true
          Initial:
          Memory: max=8232m, free=7974m, used=258m

          Added 2017 kmers; time: 0.570 seconds.
          Memory: max=8232m, free=7545m, used=687m

          Input is being processed as unpaired
          Started output streams: 0.449 seconds.
          Processing time: 118.446 seconds.

          Input: 49512700 reads 2475635000 bases.
          QTrimmed: 7288815 reads (14.72%) 218452414 bases (8.82%)
          KTrimmed: 3125475 reads (6.31%) 23413723 bases (0.95%)
          Total Removed: 4077445 reads (8.24%) 241866137 bases (9.77%)
          Result: 45435255 reads (91.76%) 2233768863 bases (90.23%)

          Time: 119.548 seconds.
          Reads Processed: 49512k 414.16k reads/sec
          Bases Processed: 2475m 20.71m bases/sec

          Comment


          • The difference is primarily because you are quality-trimming to Q27, which is too high for almost any purpose. I'd suggest a command more like this:

            Code:
            bbduk.sh -Xmx8g in=D3_464_S2_L001_R1_001.fastq.gz out=D3_464_S2_L001_R1_001_trimmed.fastq.gz ref=resources/adapters.fa threads=8 k=19 mink=5 hdist=1 hdist2=0 ktrim=r qtrim=r minlength=36 trimq=14

            Comment


            • --Hi,

              thank you for your answer, just a question about quality check:
              trimq=14 means an average quality in a sliding window such as in Trimmomatic with SLIDINGWINDOW:4:15 or not ?

              best -

              Comment


              • BBDuk supports a sliding window; the flags "qtrim=w,4 trimq=15" will give similar behavior to Trimmomatic. But I don't recommend that; the Phred trimming method used by default is optimal, whereas sliding-window trimming is non-optimal.

                Comment


                • okay good, thanks for your help.

                  Comment


                  • I am using bbduk.sh to trim fastqs to a given length using the force trim capability. I noticed that the character # is being changed to ! in the Q score line of the trimmed fastq. I was unable to find documentation describing whether this is expected behavior. Would you be able to provide some insight into this? I ran the following command:

                    ../../tools/bbmap/bbduk.sh in=<sample>.fastq.gz out=<trimmed_sample>.fastq.gz ftr=50 ordered=t

                    Original fastq:
                    @SN1131:915:HFYN7ADXY:1:1101:21364:2052 1:N:0: CAACCACA
                    TTTCNCCACCACCACGTCGTTCTTGCGCCTCTTCTTGGCTTTCCGCTTGCGCTTGGGTATCTGGCTTGGGGGGCGGAGTGGATCCTGCTTTCTGGCGGAAA
                    +
                    @@@B#2=BFHFHHII<GHIIIHIIIBHIIIIIIIIIIIIGIIIGIIIIIIIHHEEEB;C@CDDCCCBBBBBB>BBBBBB?BCCCCCCCCCCCCCCCB<9>B


                    bbduk.sh output:
                    @SN1131:915:HFYN7ADXY:1:1101:21364:2052 1:N:0: CAACCACA
                    TTTCNCCACCACCACGTCGTTCTTGCGCCTCTTCTTGGCTTTCCGCTTGCG
                    +
                    @@@B!2=BFHFHHII<GHIIIHIIIBHIIIIIIIIIIIIGIIIGIIIIIII


                    Thank you,
                    Brian

                    Comment


                    • Hi Brian,

                      That's intentional. The 5th base call is an N, which means the quality score should be 0 (!) not 2 (#). Some versions of Illumina software have bugs causing some Ns to be assigned quality scores above 0, or called bases to be assigned a quality score of 0. Neither of these cases should happen as they are mathematically contradictory, and can cause problems with downstream tools, so BBDuk automatically fixes both of them.

                      You can add the flag "changequality=f" to disable this behavior, but I don't recommend it.

                      Comment


                      • That makes sense. Thank you.

                        Comment


                        • This behaviour is a bit un-Unix like?


                          bbduk.sh in1=R1.fq.gz in2=R2.fq.gz loglog loglogk=31 out=/dev/null

                          Unspecified format for output /dev/null; defaulting to fastq.

                          Exception in thread "main" java.lang.AssertionError: /dev/null already exists; please delete it.

                          Comment


                          • Originally posted by Torst View Post
                            This behaviour is a bit un-Unix like?
                            @Brian will have a more official answer but BBTools are pure Java and are coded to be OS agnostic (will run on any OS with Java).

                            Not specifying an "out" option with most BBTools produces all statistics without result output (giving you out=/dev/null effect).

                            Comment


                            • Haha

                              The syntax would be:

                              bbduk.sh in1=R1.fq.gz in2=R2.fq.gz loglog loglogk=31 out=stdout.fq > /dev/null/

                              But, you don't need to specify anything, as the default is to not print anything rather than writing to stdout, so just do this:

                              bbduk.sh in1=R1.fq.gz in2=R2.fq.gz loglog loglogk=31

                              Edit: @Genomax beat me by a minute

                              Comment


                              • Getting different results with bbduk command line vs geneious plugin

                                Hi,

                                I have been searching the default settings in the command line and still haven't identified the source of the discrepancy... Here is my linux command:

                                sh ~/bbmap/bbduk.sh in1=~/path/to/forwards.fastq.gz in2=~/path/to/reverses.fastq.gz out=~/path/to/output.fastq.gz ref=~/bbmap/resources/adapters.fa ktrim=r k=23 mink=11 hdist=1 minoverlap=24 tbo

                                result: 3628348 reads, 581467350 bases

                                and here is my plugin command from the geneious output:
                                java.exe -ea -Xmx100m -cp ...\currenjgi.BBDukF ktrim=r k=23 hdist=1 edist=0 mink=11 ref=adapters.fa minlength=10 trimbyoverlap=t minoverlap=24 qin=33 in=input1.fastq in2=input2.fastq out=output1.fastq out2=output2.fastq

                                result: 3628348 reads, 584214527 bases

                                the plugin command seems compatible with my data and the defaults in bbduk.sh. Any idea why 3M more bases in the plugin?

                                Thanks, Aaron

                                Comment

                                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
                                7 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, Yesterday, 06:07 PM
                                0 responses
                                7 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-22-2024, 10:03 AM
                                0 responses
                                49 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-21-2024, 07:32 AM
                                0 responses
                                66 views
                                0 likes
                                Last Post seqadmin  
                                Working...
                                X