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Old 07-27-2017, 09:53 PM   #11
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,182
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1-2ug input is recommended for shotgun libraries that are sequenced deep and should have high diversity. Amplicon libraries can be sequence deep but they are low diversity so input can be reduced to 100ng and also library prep can be scaled down to reduce costs.

Generally amplicon sequencing with Illumina systems can utilise following methods:
1- Fusion primers which include target specific sequences+ linker+ padding+ index+ flow cell binding domain such as http://press.igsb.anl.gov/earthmicro...standards/16s/
2- Two step PCR as demonstrated in Illumina 16S protocol
3- Ligation of amplicons (which can include sample specific barcode at 5' end but potentially will affect quantitative results) with indexed adapters

For low diversity libraries various methods can be adapted to increase base diversity during sequencing. For qualitative application their effect would be minimal but for quantitative assays such as 16S microbial profiling diversity addition methods will affect the results.
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