Poor quality of Forward vs Reverse reads

User Support
#1

Hello,

I imported pair-ends reads (PE300, Illumina HiSeq 4000 PE100, fastq files with quality at 33bp) with the manifest option reporting R1 as Forward and R2 as Reverse reads in 2 separeted column on top of the sample-id column.
qiime tools import
–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path manifest.tsv
–output-path demux-paired-end.qza
–input-format PairedEndFastqManifestPhred33V2
Manifest:

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When I look at the demux-summay, I end it up with better quality of my reverse compared to forward reads. Any idea why? Should not be the contrary ?

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#2

Hi @Nicheca1,

this may be anecdotal but I receive very similar quality plots for my sequence reads when I analyse mixed orientation sequences.

Cheers,

Lukas

2 Likes
#3

Interesting @lukasbeule , is it maybe something not abnormal - But how explain?

#4

:thinking: I don’t know why the quality behaves like this. Maybe someone else in the forum knows the answer to it?!

#5

I would not worry about it… it’s true that reverse reads typically have lower quality than forward, but this is not a rule :man_shrugging:

Someone on this forum (can’t find the post) recently reported that their sequencing company mislabeled the forward and reverse reads, so it’s also possible :man_shrugging: worth quickly inspecting the reads before importing to see if the orientations are clearly reversed (e.g., if primers are still in the reads or if there are other patterns to search for)

Good luck!

2 Likes
#6

Hi @Nicholas_Bokulich, many thanks for your reply.