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MiSeq Fastqc - Drop in the first position

Hi everyone,

I obtained some samples of amplicon sequencing (16s, V4) from a sequencing facility where they have trimmed the adapters & etc, "ready to use". However, I was wondering why, in the fastqc report, I would have the initial drop of quality (5 bps), as it's in the figure:

fastqc per base sequence quality

Is there a technical explanation for this?

Thanks in advance

amplicon sequencing fastqc miseq
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written by andrebolerbarros • andrebolerbarros

1 answer

In your case a "technical explanantion" is likely the same sequence at the beginning of each fragment/amplicon. Illumina sequencers are designed with expectation of an equal distribution of A/C/G/T at any sequence position/cycle. Having low nucleotide diversity (all clusters glowing the same color) affects the Q scores resulting in a drop. This is the reason phiX is recommended as a spike-in with amplicons to normalize the distribution of nucleotides.

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written by GenoMax GenoMax

Thanks GenoMax! In that scenario, would that mean that, by looking at the overrepresented sequences, it should have the same 5 bp start in all of them right? But that is not what happens...

enter image description here

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written by andrebolerbarros • andrebolerbarros

Is there a particular reason you are concerned? Phred score is > Q30 for those bases.

That said 50+% of your reads have the same sequence.

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written by GenoMax GenoMax

Just being sure that it is nothing wrong with the samples. Thanks!

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written by andrebolerbarros • andrebolerbarros

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