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Old 07-08-2018, 09:16 PM   #1
johnathanlee
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Location: hn

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Default I7 index reading issue

We are working on custom designed assay for sequencing using Miseq V2 kits 300 cycles.

Following design we have used

Round 1 PCR
F-Primer
5 ACACTCTTTCCCTACACGACGCTCTTCCGATCT-[locus specific sequence]

R-Primer
5 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-[locus specific sequence]


Round 2 PCR
i5-

5'-AATGATACGGCGACCACCGAGATCTACAC-index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'

i7-

5'-CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'

We are facing problem while demultiplexing. Along with the main i7 index read multiple other i7 index reads are getting paired against same i5 read. This decreases the overall coverage when files are demultiplexed using both i7 and i5. However when files are demultiplexed using only i5 reads the file coverage increases significantly.

Is it known problem with illumina platform?
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Old 07-08-2018, 11:56 PM   #2
t.m.wasiak
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Default

Hi,

Have you every try to use opensource Galaxy software?
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Old 07-09-2018, 01:27 AM   #3
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

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Default

If they have been sequenced on MiSeq, you could have cross contamination among i7 primers.

1- It could have happen during primer synthesis where yuo will get more of other i7 index from a previous one on your oligo order list than the other ones.

2- Cross contamination during work with oligos in the lab
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