Unindexed reads in MiSeq

[dapen...@gmail.com]

Hi all,

I did a Gecko library screening following the Julia Joung Nature Protocol paper, but the MiSeq showed that most of the sequences are "unindexed reads". 

My primers are the same with the ones in the protocol, and the barcodes are shown in red below:

Gecko_R2_A	CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCGCCTTGtctactattctttcccctgcactgt
Gecko_R2_B	CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATAGCGTCtctactattctttcccctgcactgt
Gecko_R2_C	CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAAGTtctactattctttcccctgcactgt

I realized that the barcodes are not default illumina barcodes so I put these sequence as “Custom” indexes. However, although the Miseq reads look good, either the barcode sequences or the reverse complementary sequences were not found in the results. Has anyone seen this before?

Many thanks,

Dapeng.

[Julia Joung]

Hi Dapeng,

Could you take a look at the sequences in your undetermined index files to see which index sequences you are seeing? Sometimes there may have been a contamination, or too much PhiX used that may result in most sequences being unindexed reads.

Best,

Julia

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[xiaohong zhao]

Hi, Do you figure our the reason for no index reads? We recently finished the sequencing for our experiment using Nextseq, and had the  similar problem with " nearly half of the sequence reads were not demultiplexed because the index read came out as GGGGGGGG ,"

for our sequencing, it was under clustering, which made the PhiX went up to 40%. BTW, The other half of the reads are fine.

I was just wondering if there's any other possible problem with the lib prep. Will put increase lib concentration and use less PhiX  fix this problem?

Thank you very much and looking forwards to hearing from you,

Xiaohong

[赵晓鸿]

We checked the indexed and inindexed reads of our results. It looks like all the unindexed reads map to PhiX. We think it is the high PhiX causes the problem.

Thank you and hope this is useful information for people have similar issue.

On Mar 31, 2021, at 12:11, xiaohong zhao <zhaoxiao...@gmail.com> wrote:



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