Index 1 read (i7) - Low Intensity & Low Quality??

EcneuqeS · 12-12-2018, 02:16 AM

Good day,

Can anyone advise me on the following regarding a recent amplicon sequencing run on the Illumina MiSeq:

What would be the cause for a relatively low intensity and low quality Index 1 read (i7) compared to Read 1, Index 2 (i5) and Read 2?

The nucleotides at each position within the indices used were balanced.

The run was quite under-clustered.

Also, other than potential dilution problem, what are other reasons for obtaining a significantly higher alignment of Phix compared to what was originally spiked?

Thanks!

luc · 12-12-2018, 06:11 PM

Did you use custom sequencing primers for any of the reads?

MMG9 · 05-03-2019, 02:02 PM

What if this person did use custom sequencing primers? How would that effect the run and data quality?

JoeKutch · 05-06-2019, 07:43 AM

I think what Luc was considering is that if the custom sequencing primer didn't anneal properly/as efficiently as the standard Illumina primer, you would have fewer clusters primed and thus fewer clusters producing signal intensity.

If the custom primers were spiked into the Illumina primer mix, then that could also explain the higher percentage of PhiX aligning, as the PhiX clusters primed normally while the amplicon clusters did not. This is applicable to read 2 and read 1.

A low quality in the index 1 read could be explained by PhiX as well. If 30% of your clusters are PhiX, that 30% will produce no signal in index 1 and reduce your %Q30 for that read accordingly.

Index 2 can't be explained by this to my knowledge, as that primer cannot be custom.

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12-12-2018, 02:16 AM	#1
EcneuqeS Junior Member Location: South Africa Join Date: Jul 2018 Posts: 2	Index 1 read (i7) - Low Intensity & Low Quality?? Good day, Can anyone advise me on the following regarding a recent amplicon sequencing run on the Illumina MiSeq: What would be the cause for a relatively low intensity and low quality Index 1 read (i7) compared to Read 1, Index 2 (i5) and Read 2? The nucleotides at each position within the indices used were balanced. The run was quite under-clustered. Also, other than potential dilution problem, what are other reasons for obtaining a significantly higher alignment of Phix compared to what was originally spiked? Thanks!

12-12-2018, 06:11 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	Did you use custom sequencing primers for any of the reads?

05-03-2019, 02:02 PM	#3
MMG9 Junior Member Location: Wisconsin Join Date: Jan 2019 Posts: 2	What if this person did use custom sequencing primers? How would that effect the run and data quality?

05-06-2019, 07:43 AM	#4
JoeKutch Member Location: Philadelphia Join Date: Oct 2016 Posts: 17	I think what Luc was considering is that if the custom sequencing primer didn't anneal properly/as efficiently as the standard Illumina primer, you would have fewer clusters primed and thus fewer clusters producing signal intensity. If the custom primers were spiked into the Illumina primer mix, then that could also explain the higher percentage of PhiX aligning, as the PhiX clusters primed normally while the amplicon clusters did not. This is applicable to read 2 and read 1. A low quality in the index 1 read could be explained by PhiX as well. If 30% of your clusters are PhiX, that 30% will produce no signal in index 1 and reduce your %Q30 for that read accordingly. Index 2 can't be explained by this to my knowledge, as that primer cannot be custom.