Question about the emulsion-PCR: in the illustration, there is only ONE single DNA-strand which is not linked to the bead. That would mean: 60 cycles -> only 60 copies. Do they additionally use primers for the red adapter to get more copies?
Some times when sequencing on an ion torrent machine, there is a barcode that takes up a lot of data space, causing the sample data to be insufficient for analysis. But still on this Chip sequencing, re-process the chip: wash the chip, then denature again with primer sequencing and wash the chip again and add the sequencing enzyme and then run the sequencing again, then the No barcode is almost reduced. . Is there any way to reduce the No barcode at the beginning of sequencing after loading the denatured sample onto the sequencing chip?
Hi, great explanation! The drawings are also very didactic. Congrats! There is just one thing I didn't capture. How does just one strand of DNA library bind to one bead? How this specificity is reached wheter the strands receive the same adaptors? Since now, grateful.
There is way more beads than DNA strands. That way, statistically, beads only have one or none DNA strands, before amplification. After amplification, by a process unknown to me, they then filter the beads as to only keep those with DNA on them
Krishnendu Kundu Hello! Because you will only release one of the four nucleotides into the well at a time. If there is a voltage change detected, it means it was incorporated. If there’s no voltage change, that nucleotide wasn’t incorporated, so it needs to be washed out before trying the next nucleotide.
@@BasicBiochem Hello! Thank you for the great explanation. Given that it is not uncommon for the wrong base to be incorporated during DNA replication, resulting in mutations due to a higher relative ratio of bases, how can we account for this during ITS? Many thanks in advance.
@@Mishomtp I'm just a nobody, but that would require all the high fidelity polymerases on a single bead to all incorporate the wrong base at the same time in order to release enough hydrogen ions to trip the sensor. Not likely at all, given how low the error rates of high fidelity polymerases are and how many of them would have to make an error at the same time.
