Low quality for index1 (i7) but not for read2

[Walid]

Hi,
I am sequencing libraries on MiSeq (V2 and V3) and facing constant problems with my i7 reads. At the end of my read1, the quality drops and remains low during my i7 sequencing (16 cycles).
However, the quality of my read2 (which initiates from the same read2 sequence-Nextera Read2 sequencing primer- as for i7) is perfect.

My question is: how can you get good quality for read2 but bad quality for i7, given that the sequencing primer is the same? and is anyone else facing the same problem?
Thanks!
Best,

Walid

[GenomicSeq]

Walid

What does it look like for your i5 read? And can you explain the full read layout for the index reads and read 1 and 2?

Lastly, what are you sequencing and how many samples are you sequencing at a time?

[Nhung Nguyen]

Hi,

For MiSeq, primers sequencing for I1 and I2 are different. You can refer to this link for more detail information.
Indexed Sequencing Overview Guide (15057455) (illumina.com)

I have same experience with my customer when they choose wrong index i1 for sequencing. The index i1 were low-diversity and the Qscore was drop out. I have attached "Index Adapter Pooling Guide" for your reference.
Introduction (illumina.com)

On the other hands, the I1 sequencing primer in cartridge has issue, even it very rare, you should check with the seller .

Hope that it help!

[Walid]

GenomicSeq My i5 is perfect (95% >Q30)
My construct is: P7_Barcodes_Read2Nextera_UMI_Insert_Read1TruSeq_Index_P5
All reads, except for i7 (16N barcodes), have high qualities.

I have this problem whether I'm pooling libraries or sequencing only one.

Nhung Nguyen Thanks for replying. I have been facing this problem for a while with V2 and V3 kits, so I don't think it's a cartridge issue. As for the low-diversity i7, it is 16N barcodes and I should have around 100k different barcodes.

Attached is a screenshot of the illumina sequencing analysis viewer of my last run