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Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?

From the manual of Velvet: it must be an odd number, to avoid palindromes. If you put in an even number, Velvet will just decrement it and proceed. the palindromes in biology are defined as ...
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14 votes
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Fast way to count number of reads and number of bases in a fastq file?

It's difficult to get this to go massively quicker I think - as with this question working with large gzipped FASTQ files is mostly IO-bound. We could instead focus on making sure we are getting the ...
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13 votes

Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?

To expand on the answer above, in case it isn't clear, we show: Why palindromic sequences must be even in length Why palindromic sequences induce self-loops in a de Bruijn graph Why self loops in a ...
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  • 233
12 votes

QC measures for NGS sequencing

MultiQC can merge all your different reports into a single one. Which could be useful once you manage to know which QC tools to use.
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  • 129
12 votes
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How do PCR duplicates arise and why is it important to remove them for NGS analysis?

In any scenario where depth of coverage is an important factor, PCR duplicates erroneously inflate the coverage and, if not removed, can give the illusion of high confidence when it is not really ...
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11 votes
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PCA vs tSNE in single cell RNA-seq

tSNE often offers better visual representation (separation) on such complicated data than PCA. As Micheal pointed out, computing a tSNE embedding over 20.000 gene dimensions is computationally ...
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10 votes
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What is deep sequencing?

I found a post useful for this topic. It explains the difference of coverage and depth. It also has a useful explanation on how to calculate coverage and depth. Here is a copy of what the link says ...
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  • 216
9 votes

Designing a lab NGS file database schema

The Global Alliance for Genomics and Health has been working on the issue of representing sequencing data and metadata for storage and sharing for quite some time, though with mixed results. They do ...
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  • 429
9 votes

What is deep sequencing?

There are several questions in your post I'll try to answer each one: Is there any way to calculate how deep the sequencing is ? See gringer's answer. TLDR: The depth of the sequencing is how many ...
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9 votes

Delete all 4 lines of a fastq read from a fastq file using read ID

Don't do it: Your FASTQ file is either malformed or your FASTQ record spans more than four lines, which is allowed in FASTQ. For a detailed description of what can go wrong in FASTQ parsing see for ...
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9 votes
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How to count the number of mapped read in 100-bp window from a BAM/SAM file

one-liner Here's a gritty one-liner to count the number of reads in a region if you have just one region that you want to investigate. Change the 1 in ...
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  • 3,071
8 votes

What is deep sequencing?

Sequencing depth is typically calculated as the number of total bases sequenced divided by the number of bases in the target genome. An Illumina sequencing run with 2x125 bp reads and 500 million read ...
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8 votes
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How can I count the number of reads that support a variant in a bam file?

If you don't mind a bit of manual counting, then samtools mpileup -f reference.fa -r chr22:425236-425236 alignments.bam will produce output where you can count the ...
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7 votes
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Why is bwa-mem the standard algorithm when using bwa?

bwa mem is newer, faster, and [should be] more accurate, particularly for longer reads. From the bwa man page (presumably in ...
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7 votes

QC measures for NGS sequencing

We routinely run both FastQC and FastQ Screen on all of our raw sequencing reads. FastQ Screen is a tool for detecting cross-species contamination. MGA is another similar tool. There are then lots of ...
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  • 271
7 votes

How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?

Several papers have made this distinction, and a few indeed use different terms to distinguish between them. For example, Kazaux et al. (2016) acknowledge that: These constraints favour the use of ...
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7 votes

How to simulate NGS reads, controlling sequence coverage?

I am working on a Illumina sequencing simulator for metagenomics: InSilicoSeq It is still in alpha release and very experimental, but given a multi-fasta and an abundance file, it will generate reads ...
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7 votes

How do PCR duplicates arise and why is it important to remove them for NGS analysis?

PCR polymerases introduce errors. When an error arises in the first few cycles of amplifications, it will appear in a reasonably high fraction of DNA fragments in the library. After sequencing, you ...
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6 votes
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How can I call structural variants (SVs) from pair-end short read resequencing data?

I think the best method or combination of methods will depend on aspects of the data that might vary from one dataset to another. E.g. the type, size, and frequency of structural variants, the number ...
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6 votes

Fast way to count number of reads and number of bases in a fastq file?

The following is more than twice as fast; however, wc counts newline characters as well. We thus need to subtract the line count from the base count (using Bash): <...
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6 votes

Fast way to count number of reads and number of bases in a fastq file?

pigz | awk | wc is the fastest method First off for benchmarks with FASTQ it's best to use a specific real-world example with a known answer. I've chosen this file: ftp://ftp.1000genomes.ebi.ac.uk/...
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  • 1,029
6 votes
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FASTQC overrepresented sequences after trimming

As @AaronBerlin mentioned, you didn't remove reads that were completely trimmed. Next time use the --minimum-length option and set it to something reasonable, like ...
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  • 19.2k
6 votes

Delete all 4 lines of a fastq read from a fastq file using read ID

If you are 100% sure the read only has 4 lines (they can have more), you can use this sed command: ...
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  • 8,111
5 votes

QC measures for NGS sequencing

The quality control of ngs reads is heavily dependent on type of the project. For genome assembly projects based on short reads, beside already covered checking quality of sequencing, you would like ...
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  • 5,287
5 votes

Is there a point in recalibration of scores for variant calling?

I personally don't think BQSR has a huge impact on variant calling, but you don't really need to guess. If you run GATK BQSR, it outputs a table and charts of exactly how much quality scores are ...
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5 votes
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Designing a lab NGS file database schema

For metadata, I would use a SQL schema something like the following: ...
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  • 5,735
5 votes

Designing a lab NGS file database schema

I agree that there is no ideal data model that is going to be stable for very long in a quick-moving field like genome informatics. Perhaps a schema-less (NoSQL or some other document-based system, ...
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5 votes

How can I use annotations to remove variants not relevant to cancer risk?

While your question is specific to cancerous germline mutations, I'd suggest you look at the COSMIC database of somatic mutations to include in your analysis. There are other factors to include in ...
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5 votes

What is the difference between NGS, 2GS, SBS and HTS?

Here are my attempts at definitions: Sanger: A method of sequencing that depends on chain-terminatiing dideoxynucleotides. This sequencing uses the differential flow of DNA sequences of different ...

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