I am independently working on data retrieved SRA database, paired-end data as separate inputs. The proceedure I followed is,

  1. After running FastQC using Galaxy, the majority of the modules have failed.
  2. I tried Trimmomatic with default settings (Avg quality =20, number of bases to avg across = 4), it resulted in R1 and R2 paired and unpaired (4 total outputs).
  3. I ran FastQC again, which rendered some changes (per base sequence quality passed) but resulted in other modules (Sequence length distribution and per tile quality scores) getting a warning sign or failing.

My question is,

  • Is there specific setting other than default that provides better results? What output should i use from Trimmomatic for fastqc again?
  • $\begingroup$ Hey, could you please be more specific about your data? What type of sequencing technology has been used? And what exactly are you trying to achieve? Are you trying to make all FastQCs modules pass the test ? $\endgroup$
    – user324810
    Commented Dec 16, 2020 at 15:25
  • 1
    $\begingroup$ Also note that, depending on your data, you might not WANT all fastqc checks to pass. If you're using 16s amplicons of a limited set of different microbes you will always get fastqc warnings on some checks due to the nature of the data. $\endgroup$
    – Pallie
    Commented Dec 16, 2020 at 16:57
  • $\begingroup$ Thank you! its Illumina MiSeq paired end sequencing. I figured not all QCs can pass in this dataset. I am still confused to what output I have to move forward with the alignment? $\endgroup$ Commented Dec 22, 2020 at 11:07


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