smaller fragments then cut out after gel purification

susch · 03-06-2011, 08:40 AM

hi there,

can anybody explain me, why i become in pcr 300 bp fragments, but cut out in the gel after ligation of the adapters 400-450 bp?
in NEBNext sample prep i see the problem and for the truseq protocol illumina gives warning for this:

"Cutting a band between 400–500 bp will result in an insert size of approximately 300–400 bp, accounting for the size of the adapters. Adapters add approximately 120 bp to each fragment. The sequencing read length should be considered when cutting fragment sizes. Sequencing reads that over‐reach into the adapter will cause chimeric reads, unalignable to the reference sequence."
is there a name for this? is the effect only for ds DNA Fragments?

upenn_ngs · 03-07-2011, 06:22 AM

the sequencing adapters add ~60bp to either end of the fragments gDNA (referred to as the "insert"). for 2x100bp sequencing, an insert size of 300bp (+120bp adapters =420bp total) ensures that the data collected from read 1 and read 2 will not overlap. for example if the insert size were 150bp, then with 2x100bp reads, 50bp from each fragment would be duplicate data. i would file this under duplicate bases (vs. duplicate reads, which are largely pcr artifacts)

that said, I believe GATK can handle data with base calls in the adapter sequence. however, i am unsure whether this sequence is trimmed or if the read is discarded.

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03-06-2011, 08:40 AM	#1
susch Junior Member Location: berlin Join Date: Jan 2011 Posts: 2	smaller fragments then cut out after gel purification hi there, can anybody explain me, why i become in pcr 300 bp fragments, but cut out in the gel after ligation of the adapters 400-450 bp? in NEBNext sample prep i see the problem and for the truseq protocol illumina gives warning for this: "Cutting a band between 400–500 bp will result in an insert size of approximately 300–400 bp, accounting for the size of the adapters. Adapters add approximately 120 bp to each fragment. The sequencing read length should be considered when cutting fragment sizes. Sequencing reads that over‐reach into the adapter will cause chimeric reads, unalignable to the reference sequence." is there a name for this? is the effect only for ds DNA Fragments?

03-07-2011, 06:22 AM	#2
upenn_ngs Member Location: philadelphia Join Date: Sep 2009 Posts: 70	the sequencing adapters add ~60bp to either end of the fragments gDNA (referred to as the "insert"). for 2x100bp sequencing, an insert size of 300bp (+120bp adapters =420bp total) ensures that the data collected from read 1 and read 2 will not overlap. for example if the insert size were 150bp, then with 2x100bp reads, 50bp from each fragment would be duplicate data. i would file this under duplicate bases (vs. duplicate reads, which are largely pcr artifacts) that said, I believe GATK can handle data with base calls in the adapter sequence. however, i am unsure whether this sequence is trimmed or if the read is discarded.