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  • Did my sequencing center did a sloppy job?

    Hello, I have just got my 16S rRNA amplicon sequencing back. We use typical Earth Microbiome Project primers 515F-806R, which is about 290bp size of amplicon.

    We requested MiSeq 300bp X 2 pair-end sequencing. We got to files back R1.fastq and R2.fastq. I just checked these 2 files. The average of raw reads in these two files is 150 bp?

    Did my sequencing center do a sloppy job? or they used wrong chemistry, kit?

    I suppose my forward read and reverse read can be as long as to 290 bp, if they do 300bp X2?

  • #2
    The average of raw reads in these two files is 150 bp?
    What does that mean? Is the data pretrimmed, which is why you have much shorter reads than expected? If all reads are 150 bp, sequencing facility may have made an error with run setup, if you had requested 2 x 300 bp.

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    • #3
      I'm curious why you would select 2x300 for a 290 bp amplicon. Did you want to merge the paired reads for extra-high quality sequence?
      Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

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      • #4
        Originally posted by SNPsaurus View Post
        I'm curious why you would select 2x300 for a 290 bp amplicon. Did you want to merge the paired reads for extra-high quality sequence?
        I am sorry. I am not sure if I understand you.

        These days MiSeq 300bpX2 is popular (default) and long reads also better for downstream analysis such as taxonomic assignment . It doesn't matter what you amplicon length is. In my case, I don't have to join them. I can choose only use Forward sequencing data (normally better than reverse sequencing data). Or I can choose to use F and R without joining or joining.

        Can you tell me what you would select? if you don't select 2X300bp?

        Comment


        • #5
          Originally posted by GenoMax View Post
          What does that mean? Is the data pretrimmed, which is why you have much shorter reads than expected? If all reads are 150 bp, sequencing facility may have made an error with run setup, if you had requested 2 x 300 bp.
          "What does that mean?" -- This is new sequencing center that I recently changed. It's first time that I used. "sloppy work" means they didn't do a good job and something wrong about the sequencing chemistry and sequencing process.

          "Is the data pretrimmed". I doubt it is pre-trimmed. They only gave me 2 files. One forward fastq file and one reverse fastq file. If they have do some trimming or analysis, sequencing center would give you 4 files another two files would be F and R fastq files after trimming.

          "If all reads are 150 bp, sequencing facility may have made an error with run setup" -- Yes, all reads are 150bp. When you said made an error, do you mean their setting wrong? for example, 2X300bp should run 300 runs. they only run 150 cycles? The machine suddenly stopped or wrong settings. Or do you mean they use wrong prep kits? for example, Hiseq commonly does 2X150bp or 2X250 bp? They use hiseq prep kits?
          Last edited by SDPA_Pet; 07-12-2019, 01:14 PM.

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          • #6
            Our local facility offers 2x300 v3 MiSeq runs with 25 million reads and a 2x150 v2 run with 15 million reads. If I didn't need the extra 10 million reads, I might be tempted to just do 2x150 and merge the two reads, and save 40% on the run costs. Did it look like you got a v3 or v2 run, based on the number of reads (or any other info)?

            It sure sounds to me like they chose the wrong read length but maybe you can still use the data? I always find it is most important in these situations to figure out (with them) where the process went wrong to help in the future. Maybe they need to formalize how the runs are requested--if it is just an e-mail and some people say paired-end 300 bp (meaning total length of 300 bp) and others paired-end 300 bp (meaning 2 reads of 300 bp each) then they rely a key bit of info that is open to mis-interpretation.
            Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

            Comment


            • #7
              Originally posted by SNPsaurus View Post
              Our local facility offers 2x300 v3 MiSeq runs with 25 million reads and a 2x150 v2 run with 15 million reads. If I didn't need the extra 10 million reads, I might be tempted to just do 2x150 and merge the two reads, and save 40% on the run costs. Did it look like you got a v3 or v2 run, based on the number of reads (or any other info)?

              It sure sounds to me like they chose the wrong read length but maybe you can still use the data? I always find it is most important in these situations to figure out (with them) where the process went wrong to help in the future. Maybe they need to formalize how the runs are requested--if it is just an e-mail and some people say paired-end 300 bp (meaning total length of 300 bp) and others paired-end 300 bp (meaning 2 reads of 300 bp each) then they rely a key bit of info that is open to mis-interpretation.
              Hmm, interesting. I didn't realize MiSeq has 2X150bp runs choice. Again, this is new sequencing center. My previous center for Amplicon sequencing only has 2X300bp or default is this settings. I am not sure if I could use this data.

              As you know my amplicon is about 290 bp. If I use 300bpX2, it perfectly covers the whole amplicon twice (F and R). However, if it is 150bp X 2, even if I could join F and R reads together, it only has <50bp overlapped fragment, it won't join very well. If I don't join this, I only have 150 bp information and I lost half of them. Why would I originally brother to choose 290 size amplicons.

              As you know, it is not Shortgun genomics, which shorter reads or longer reads may have different advantage in assembly etc.

              Comment


              • #8
                @SDPA_Pet: Your sequencing facility may have simply run 2x150 sequencing by mistake if you are sure the reads have not been trimmed.

                You should be able to ask them to re-sequence the sample 2x300, as originally requested at no charge, if they made that error.

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                • #9
                  Maybe they used transposase tagmentation, rather than directly ligating adapters onto the input DNA.

                  Comment


                  • #10
                    Perhaps there just was a mistake/typo during the demultiplexing/bcl2fastq conversion. Just talk to your facility.

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                    • #11
                      You should definitely call them.

                      Someone probably was in a hurry and loaded a 300 cycle kit after seeing 300 paired end, when they should have loaded a 600 cycle. They should be able to easily check which kind of kit they loaded onto the sequencer. Could be many things, but I have definitely seen that mistake before.

                      Comment

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