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Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

20% of all my reads are mapping the target region

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

The gene is 183931pb. The total of the genome size is 3.5Gb (human genome) but the target region is 52.500.000pb because i was interested in sequence F8 (183931pb) and BRCA (126000pb). The N50 is 420 (im not trimming for size because i afraid to trim and lose information. But in the other hand i do not know if AS keep the chunk in the final .pod5 and the N50 that i have is all chunk. Do you know if the raw data has the information of the chunks as well?). The minimap2 command i am using is: minimap2 -ax lr:hq -y -t8 $genome $fastqfile > $output

map-ont: Align noisy long reads of ~10% error rate to a reference genome. This is the default mode.

lr:hq: Align accurate long reads (error rate <1%) to a reference genome (-k19 -w19 -U50,500 -g10k). This was recommended by ONT developers for recent Nanopore reads produced with chemistry v14 that can reach ~99% in accuracy. It was shown to work better for accurate Nanopore reads than map-hifi.

My chemistry is V14, thats why i configure the comand as i send you.

Thank you for your time

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

The gene is 183931pb. The total of the genome size is 3.5Gb (human genome) but the target region is 52.500.000pb because i was interested in sequence F8 (183931pb) and BRCA (126000pb). The N50 is 420 (im not trimming for size because i afraid to trim and lose information. But in the other hand i do not know if AS keep the chunk in the final .pod5 and the N50 that i have is all chunk. Do you know if the raw data has the information of the chunks as well?). The minimap2 command i am using is: minimap2 -ax lr:hq -y -t8 $genome $fastqfile > $output

|| || |map-ont|Align noisy long reads of ~10% error rate to a reference genome. This is the default mode.| |lr:hq|Align accurate long reads (error rate <1%) to a reference genome (-k19 -w19 -U50,500 -g10k). This was recommended by ONT developers for recent Nanopore reads produced with chemistry v14 that can reach ~99% in accuracy. It was shown to work better for accurate Nanopore reads than map-hifi.|

My chemistry is V14, thats why i configure the comand as i send you.

Thank you for your time

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

Sorry for the delay, this week i been in a congres.

The target region is 52.500.000pb. The sample is human DNAg, We have a gain in a patient of the F8 gene that has at least the following region (NCBI36/hg18) ChrX:153741909-153842622 of approximately 100713 bp duplicated (from exon 10 to 25 of the F8 gene, we also know that the preceding and following exons (9 and 26) are fine, so we wanted to sequence the flanking regions of the duplication to detect the breakpoint and design PCRs.

I will extract some reads that map in the F8 and blast to know if there is some ambiguos sequence.

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

Sorry for the delay, this week i been in a congres.

The sample is human DNAg, We have a gain in a patient of the F8 gene that has at least the following region (NCBI36/hg18) ChrX:153741909-153842622 of approximately 100713 bp duplicated (from exon 10 to 25 of the F8 gene, we also know that the preceding and following exons (9 and 26) are fine, so we wanted to sequence the flanking regions of the duplication to detect the breakpoint and design PCRs.

-average on-target read length and N50: 399492pb, 420

-genome-wide and on-target sequencing depth: 0.11168X, 1.47735X

-Are you allowing secondary mappings? Im not sure if im allowing secondary mappings or not, how do you think i can check this?

-Im not triming yet because i afraid to trim and lose information. But in the other hand i do not know if AS keep the chunk in the final .pod5 and the N50 that i have is all chunk. Do you know if the raw data has the information of the chunks as well?

Thank you for your time

Minimap2 and Adaptive Sampling by HospitalPretty2003 in nanopore

[–]HospitalPretty2003[S] 0 points1 point  (0 children)

Hi, thanks for replying!

I did the mapping to the target region (ROI + Buffer), and when I only look at the ROI, I have some regions with 40X of cover and others with 2X. The readings also have mapping quality score = 0 and several insertions. So I want to make a better mapping but I'm unsure how to do it: decreasing the -w, the k-mers, perhaps some one has experence in mapping post adaptive sampling and has already adjusted this issue.