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Forum: Oxford Nanopore 05-30-2018, 05:29 AM
Replies: 1
Views: 301
Posted By gringer
Clive Brown's Technology Talk from London Calling 2018

For the last 4 years I've attended Oxford Nanopore Technologies' yearly London Calling conference. One of the interesting bits of this conference is the talk from Clive Brown about what's happening...
Forum: Bioinformatics 05-28-2018, 05:20 AM
Replies: 1
Views: 322
Posted By gringer
That should be fine. Inchworm does a kmer-based...

That should be fine. Inchworm does a kmer-based assembly, so as long as the reads are longer than the size of the kmers, it should be okay.

However, it'd be a good idea to do adapter and end...
Forum: Bioinformatics 04-22-2018, 04:31 AM
Replies: 2
Views: 332
Posted By gringer
If you don't care about accuracy, just...

If you don't care about accuracy, just concatenate all the fasta sequences into a single large sequence....

More seriously, you're probably not going to get any better continuity from your data...
Forum: Bioinformatics 04-22-2018, 04:25 AM
Replies: 13
Views: 1,412
Posted By gringer
That's not the only reason. Sigma also has...

That's not the only reason. Sigma also has region-specific pricing. Without logging in, I choose the location when I first visit the site (presumably stored as a cookie for subsequent visits), and...
Forum: Bioinformatics 04-22-2018, 03:00 AM
Replies: 13
Views: 1,412
Posted By gringer
You won't be able to get a good de-novo assembly...

You won't be able to get a good de-novo assembly from Illumina data alone, and longer SBS reads won't fix that. Consider things like 28S rRNA gene regions, where kilobase-length regions are repeated...
Forum: Bioinformatics 04-20-2018, 04:25 PM
Replies: 13
Views: 1,412
Posted By gringer
As far as I know, ONT is the only sequencing...

As far as I know, ONT is the only sequencing machine supplier that publishes all their prices on their store website; you can have a look and see for yourself!

At the moment marginal cost per base...
Forum: Bioinformatics 04-17-2018, 04:37 PM
Replies: 13
Views: 1,412
Posted By gringer
If you want cheap,...

If you want cheap, [MinION](https://store.nanoporetech.com/basic.html) is always available (starter discount is $1000 for two flow cells and a rapid kit). The consensus assemblies are less accurate...
Forum: Metagenomics 04-17-2018, 04:32 PM
Replies: 1
Views: 407
Posted By gringer
Unlikely. Sanger sequencing doesn't really work...

Unlikely. Sanger sequencing doesn't really work with variants at a frequency below about 20%, and any INDEL variation in the community is going to mess up the sequencing quite a lot.
Forum: General 04-17-2018, 04:12 PM
Replies: 2
Views: 1,027
Posted By gringer
I'd recommend doing an analysis on the unmapped...

I'd recommend doing an analysis on the unmapped reads to make sure that they are not hitting something other than your intended target. With a tumour sample it's unlikely to be a contamination issue,...
Forum: Bioinformatics 04-17-2018, 04:06 PM
Replies: 1
Views: 281
Posted By gringer
My usual approach is to do the mapping...

My usual approach is to do the mapping separately, and do variant calling in parallel with samtools mpileup in multi-BAM mode (i.e. using more than one BAM file on the command line). This increases...
Forum: Bioinformatics 02-19-2018, 09:31 AM
Replies: 3
Views: 585
Posted By gringer
Something else to try: get rid of reads that have...

Something else to try: get rid of reads that have '.' in their sequence:

http://seqanswers.com/forums/showthread.php?t=6297
Forum: Bioinformatics 02-18-2018, 02:33 PM
Replies: 3
Views: 585
Posted By gringer
I expect that you should be putting csfastq files...

I expect that you should be putting csfastq files into bowtie, rather than csfasta + qual.

But you'd be a lot better off leaving SOLiD data alone. Replicate the experiment using full-length cDNA...
Forum: Oxford Nanopore 02-04-2018, 10:36 PM
Replies: 5
Views: 1,470
Posted By gringer
I can tell you what I use, but it's unlikely to...

I can tell you what I use, but it's unlikely to be what other people prefer, because there's a lot of manual computational legwork involved.

De-novo Assembly -- Canu...
Forum: Bioinformatics 02-03-2018, 11:57 AM
Replies: 4
Views: 750
Posted By gringer
My probably-biased point of view (as a...

My probably-biased point of view (as a bioinformatician) is that even with the original CLC prices, a company would get more benefit out of hiring a dedicated bioinformatician than paying for CLC.
...
Forum: Oxford Nanopore 02-03-2018, 11:52 AM
Replies: 5
Views: 1,470
Posted By gringer
Both the command-line program ONT provides for...

Both the command-line program ONT provides for basecalling (Albacore) and the GUI Client that does sequencing and optionally basecalling (MinKNOW) can now output fastq files directly, no need to...
Forum: Oxford Nanopore 01-30-2018, 10:09 AM
Replies: 4
Views: 1,770
Posted By gringer
The costs you have stated are per sequencing run,...

The costs you have stated are per sequencing run, not per sample (i.e. $100 per run + $56 per run), so divide those numbers by six; please re-read the post that you replied to.

If bacterial genome...
Forum: Pacific Biosciences 12-18-2017, 12:03 AM
Replies: 14
Views: 2,665
Posted By gringer
As fas as I know, canu also doesn't use quality...

As fas as I know, canu also doesn't use quality scores for assembly. By default it does its own error correction based on an all-vs-all read mapping, and produces corrected FASTA files (i.e. without...
Forum: Oxford Nanopore 12-17-2017, 11:40 PM
Replies: 4
Views: 1,770
Posted By gringer
Nanopore costs are all available on the nanopore...

Nanopore costs are all available on the nanopore store website (https://store.nanoporetech.com/kits-218.html). Here are some demonstrative figures:


Flow cells at their most expensive are $900...
Forum: Oxford Nanopore 11-03-2017, 10:49 AM
Replies: 4
Views: 2,034
Posted By gringer
Nanopolish also now includes methylation in its...

Nanopolish also now includes methylation in its correction model, which (according to Ryan Wick) has brought the nanopore-only consensus accuracy up to q30 for a bacterial genome.
Forum: RNA Sequencing 10-23-2017, 12:56 AM
Replies: 2
Views: 1,169
Posted By gringer
It is a type of technical replicate, but will...

It is a type of technical replicate, but will have some degree of biological variation from the different samples. Usually "technical replicate" refers to an additional sequencing library prepared...
Forum: Bioinformatics 10-23-2017, 12:47 AM
Replies: 2
Views: 501
Posted By gringer
Should the refcount be the sum of the count of...

Should the refcount be the sum of the count of the two assemblies (i.e. 60528, not 28905)?
Forum: Illumina/Solexa 10-18-2017, 10:59 AM
Replies: 26
Views: 4,037
Posted By gringer
I was told by an Illumina rep at Queenstown...

I was told by an Illumina rep at Queenstown Research week about a month ago that NovaSeq run cost would be similar to HiSeq, but produce about double the output.
Forum: Oxford Nanopore 09-25-2017, 02:28 PM
Replies: 4
Views: 2,034
Posted By gringer
The 'ont2d' flag sets up BWA parameters to work...

The 'ont2d' flag sets up BWA parameters to work for ONT data. It was named at a time when 2D data was similar accuracy to what 1D data is at the moment. You should be fine using Albacore 2.0-called...
Forum: General 08-25-2017, 04:09 PM
Replies: 3
Views: 870
Posted By gringer
Experimental design: not choosing appropriate...

Experimental design: not choosing appropriate controls, too few biological replicates, or doing sequencing on the wrong cell population (e.g. whole-blood RNASeq for an obesity study).
Forum: Bioinformatics 08-23-2017, 03:30 AM
Replies: 4
Views: 779
Posted By gringer
Samtools mpileup has its own INDEL correction,...

Samtools mpileup has its own INDEL correction, which may be influencing the results that you're seeing. You could try skipping this (option `-IŽ) and seeing if it changes the results.
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