Hi there, could any one tell me why all the reads in my fastq file have the same or very similar quality value as showing below? Thanks a lot.
@NB502075:3:H2FM3BGX3:1:11101:9808:6370 2:N:0:TAGAACAC TTCGCCTAGTGGGAGCTTATCTTCTCGCCACCCTCGGTGGAAATGCCTCCCCATCTGCACAAGATGTCTTTAAGG + AAAAAEEE/EEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE @NB502075:3:H2FM3BGX3:1:11101:7933:6371 2:N:0:TAGAACAC TCAGGATTCTTTGAGTCTTGGATAATGGCTCCCGCTCGTAAGGGAAAGGCTAAGGAGGAACAGGCTGTCGTGTCC + AAAAAEEEEEEEEEEEEEEEEEEEEEAEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEE @NB502075:3:H2FM3BGX3:1:11101:19490:6372 2:N:0:TAGAACAC TCGTAGGTGACGGGTTTTTCCTATGCCGAAAGGTATCGGTAAACCGTTGAAATTCTTCCATGTCCGGGATAGGGT + AAAAAEEE/EEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE @NB502075:3:H2FM3BGX3:1:11101:3601:6372 2:N:0:TAGAACAC ACGTCAACTCTACGCTGGCGTAAATCAATGTTGGAAACACTCGAAGAACAACGAAAAGCCGTCGAAGGTGAAGCA + AAAAAEEE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE @NB502075:3:H2FM3BGX3:1:11101:16612:6373 2:N:0:TAGAACAC AGTGCTGTTACCATTAGGACTGACAGATTGAAAGCTCTTTCTCGATTTGGTGGTTGGTGGTGCATGGCCGTTCTT + AAAAAEEE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE
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Since this is a NextSeq machine, please note that base qualities are binned into a few discrete values in that machine. For that reason, as long as the library is reasonably good quality, you'll relatively rarely see the error bars in FastQC reach much past 1 bin (a change of 5 on the Phred scale). For comparison, I just pulled a random sample from a recent NextSeq run and see 0 variance for the first ~30 or 75 bases in FastQC. I only see a yellow box in the last base. If you have a really messy library then you'll get more variance in that plot.
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Follow up the original question, when I ran through fastqc, there is no error bar in the per base sequence quality. So the graph looks not usual.
Plase use
ADD COMMENTinstead of answer unless you are actually answering your own post.Indeed. I have now moved his answer to a comment.
Can you provide a bit more information about the sequences? What sort of machine? What library size/type of sequencing is it? Does all of your data look like this?
The machine ID is for a NextSeq ("NBXXXXXX").
Where did they come from? Straight off the machine?