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What conditions do I use to anneal the adapters and what concentration is optimal for ligation of adapters to the fragmented genomic DNA.
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What kind of experiement are you doing? de novo sequencing, exome capture, etc? you can find these information in a standard protocol ...Nicolas Tremblay
Graduate Student
Cardiovascular Genetics - Andelfinger Lab
CHU Ste-Justine Research Center -
I mapped the illumina reads (unpaired reads) using Bowtie2. Now i want to extract the uniquely mapped reads. the formate of mapping summary is...
20000 reads; of these:
20000 (100.00%) were unpaired; of these:
1247 (6.24%) aligned 0 times
18739 (93.69%) aligned exactly 1 time
14 (0.07%) aligned >1 times
93.77% overall alignment rate
So please help me if anyone know that how to extract the uniquely mapped reads..Comment
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It depends on what metrics you want to use to call something uniquely mapped. It's more useful to think of alignments in terms of probability of being correct, which which case just use something like MAPQ>=10. BTW, bowtie2 will give multimappers MAPQ scores of 0 or 1, but that doesn't mean that alignments with MAPQ scores of 2 are that reliable.Comment
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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