3/6/2015 DR 提醒
1: Daw-Yang seems to use Clontech ds-cDNA prep for single cell Seq + Tagmentation
2: There is no way to add specific primer in this way…
According to Daw-Yang: I will use Nexera XT for DNA prep.
Figure 1 AMP for targeted RNA and DNA sequencing (pdf 4231).
Double-stranded cDNA (dscDNA) synthesis starts with total nucleic acid or RNA from fresh or FFPE material without ribosomal RNA or genomic DNA(gDNA) depletion. Solid-phase reversible immobilization (SPRI)-cleaned double-stranded cDNA or fragmented genomic DNA is processed with end repair and dA tailing, directly followed by ligation with a half-functional adapter. SPRI-cleaned, ligated fragments are amplified with 10–14 cycles of multiplex PCR1 using gene-specific primers (GSP1 pool) containing a PCR multiplexing tag (brown) and a primer complementary to a portion of the universal ligated adapter (short green). An unknown fusion partner or target sequence is indicated by a question mark. SPRI-cleaned PCR1 amplicons are amplified with a second round of 10-cycle multiplex PCR2 using a combination of GSP2 pool nested gene-specific primers (3′ downstream of GSP1), which are tagged with the second adapter sequence specific for Ion Torrent (black–blue) or Illumina (black–red, subsequently tagged with an indexing primer (red-orange-purple)), and a second nested primer against the ligated universal adapter (long green). After a final SPRI cleanup, the target amplicon library is ready for quantitation, downstream clonal amplification and sequencing.
Fluidigm 4-Primer Amplicon Tagging
The Access Array 4-primer amplicon tagging scheme is based on a step-out four primer PCR strategy where tagged target-specific (TS) primer pairs are combined with sample-specific primer pairs that contain a barcoding sequence and the adaptor sequences used by Illumina sequencing systems. By incorporating sample-specific barcodes, all 2,304 PCR products generated from a 48.48 Access Array Integrated Fluidic Circuit (IFC) are unique and can be pooled together to run in a single sequencing experiment.
Nextera_xt_sample_prep