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Old 08-29-2013, 07:42 PM   #1
Location: Dallas

Join Date: Aug 2013
Posts: 13
Default Read length

Hi everyone,

I am still new to the next gen world. I have a question about read length. I am working with nextera custom enrichment, so our product is from 300 to 1000. I am still a little confused on how the cycling occurs. We are doing 150bp paired end sequencing, so what happens to the 1000bp sequences when it goes through 150 cycles? Is 700bps or so just not read? I hope this makes sense. I am just confused on how you perform say 150bp sequencing, but not exactly 150bp is in between the adapters.

Thanks in advance.
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Old 08-29-2013, 10:07 PM   #2
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Location: Eugene, OR

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You are correct, despite your confusion! You can have fragments of many sizes. Say there is a 500 bp fragment, and the run is paired-end 100 bp reads. You'll get 100 bp of the left end of the fragment, and 100 bp of the right end of the fragment, and 300 bp of unknown sequence between. If the fragment is 100 bp, you read the same DNA twice, in different directions.

The sequencing starts with a primer hybridizing to the adapter of your template. One nucleotide is added at a time, visualized for incorporation, and the process is repeated for the number of cycles desired (100 bp, 150 bp, etc.).
Providing nextRAD genotyping and PacBio sequencing services.
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Old 08-30-2013, 07:07 AM   #3
Location: Dallas

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Thanks a lot!
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Old 08-30-2013, 07:15 AM   #4
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Another small point is that the piece in between your two 100bp reads is unknown, but we know the two 100bp pieces should align 'somewhere' together. So finding that 'somewhere' is more accurate based on this, resulting in less wasted sequence following alignment.
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