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Old 01-01-2017, 08:23 PM   #1
Shimul
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Location: Brisbane

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Default Library insert size

Hi There,

I am aiming to sequence bacterial genome with NexteraXT 300bp paired-end reads. I have made my library and all the fragments size (including the adapter) are ranging from 200-650 (I check with the bioanalyzer and gel). Do you think that I will have sufficient insert size in the end? Do you think that I will have good insert size for clustering on the MiSeq platform (using Nextera v3 chemistry)?

Would be very helpful of your suggestions here!

Cheers,

Shimul
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Old 01-01-2017, 09:06 PM   #2
nucacidhunter
Jafar Jabbari
 
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Insert sizes will be around 50-500 bp and most of sequenced fragments will be from shorter inserts and their abundance will depend on size distribution of library fragments. Library should cluster and sequence fine but you will not benefit from the long reads. To sequence larger fragments library fragments should be 600-1500 bp.
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Old 01-01-2017, 10:59 PM   #3
Shimul
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Thanks a lot @ nucacidhunter
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Old 01-06-2017, 07:34 AM   #4
Shimul
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Quote:
Originally Posted by nucacidhunter View Post
Insert sizes will be around 50-500 bp and most of sequenced fragments will be from shorter inserts and their abundance will depend on size distribution of library fragments. Library should cluster and sequence fine but you will not benefit from the long reads. To sequence larger fragments library fragments should be 600-1500 bp.
Hello,

I am finally running of my sample on the Miseq using MiSeq V3. I have checked the machine now and it shows that after 70 cycles, the >=Q30 is 96%, cluster passing filter 91.4%, and the cluster density is 1092K/mm.

Do you think that the run is going well?

Hope to hear back from you soon again!

Cheers,

Shimul
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Old 01-06-2017, 07:43 AM   #5
jdk787
josh kinman
 
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Quote:
Originally Posted by Shimul View Post
Hello,

I am finally running of my sample on the Miseq using MiSeq V3. I have checked the machine now and it shows that after 70 cycles, the >=Q30 is 96%, cluster passing filter 91.4%, and the cluster density is 1092K/mm.

Do you think that the run is going well?

Hope to hear back from you soon again!

Cheers,

Shimul
Those numbers are good, so the run is probably going well.
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Old 01-06-2017, 08:31 AM   #6
Shimul
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Default jdk787

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Originally Posted by jdk787 View Post
Those numbers are good, so the run is probably going well.
Thanks a lot Josh!

Cheers,

Shimul
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Old 01-06-2017, 10:08 AM   #7
HESmith
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@Shimul, it is not necessary to post the same question multiple times on the forum. Your run metrics look good.
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