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9 votes
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What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?

tldr - The I*.fastq.gz file contains the read index sequences. long explanation Illumina uses a program called bcl2fastq to demultiplex sequencing runs. This ...
conchoecia's user avatar
  • 3,161
8 votes

relation between Illumina sequencing primer and viral sequences

You'll unfortunately find adapter sequences contaminating a lot of entries in Genbank. In short, the sequence is most likely either an unassembled fragment where the input into the assembly step hadn'...
Devon Ryan's user avatar
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6 votes

what percentage of the human genome is MAPQ=0?

Mapping quality is determined by the repetitiveness of the genome, the sequencing error rate, insert size, the capability of the mapper and the nasty heuristics behind the mapper. MAPQ=0 to one mapper ...
user172818's user avatar
  • 6,515
6 votes
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genes with highest RPKM/FPKM for a human RNA-seq experiment?

As asked as it is, the answer is probably no. Indeed, the highest expressed RPKM/FPKMs will be different from one condition (or tissue) to another one for example. Then, you may also have technical ...
Mitra's user avatar
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6 votes
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"Sequence Duplication Levels" module still fails after pre-processing Illumina data

To answer your direct question, there are a few reasons why there might be high levels of sequence duplication. From the FastQC help: The underlying assumption of this module is of a diverse ...
Daniel Standage's user avatar
6 votes

"Sequence Duplication Levels" module still fails after pre-processing Illumina data

FastQC assumes that all samples are for whole genome sequencing and will flag them as failed if they differ too much from that assumption. This will, for example, cause essentially all RNA-seq, ChIP-...
Devon Ryan's user avatar
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6 votes
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How to align output of grep --color=always? (To QC fasta/fastq files)

Perhaps, grep is not the best tool to use in this case, but it should be in principle possible by using grep & ...
Iakov Davydov's user avatar
6 votes

How much does Nanopore cDNA Sequencing Cost?

The standard PCR-cDNA barcoding kit is \$750 USD, and has enough reagents for six runs, with each run using up to 24 samples (i.e. 144 samples total). Each run also needs one MinION flow cell (\$900 ...
gringer's user avatar
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5 votes
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Why do NEBNext indexing primers have sequence between the p5 oligo and index?

Question 1: The additional sequences is needed because that is complimentary to the P5 sequence anchored to the flow cell. It is also the site for priming the Index 2 read on a MiSeq. The additional ...
Beth Nelson's user avatar
5 votes
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Analyzing Illumina Counts

The counts files for GSE89225 is the output of HTSeq-count as a large matrix. Unless you are developing a differential expression package yourself you should not attempt to directly use this. Rather, ...
Devon Ryan's user avatar
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5 votes

what percentage of the human genome is MAPQ=0?

As has been said before, mappability to the 'human genome' depends on a number of factors, among these the reference version and type of reads, for which you are interested in GRCh38 and 2x150bp reads....
JRodrigoF's user avatar
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5 votes

SNP located within a promoter region (pig)

As far as I'm aware, Illumina provide CSV annotation files for all their sequencing chips, which can be used when they can't be found in Bioconductor. You can find annotation information for the ...
gringer's user avatar
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4 votes
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Does the MAPQ=0 fraction of a BAM file depend on the insert sizes?

Yes, as a general rule of thumb mappability increases with insert size (up to a limit) and read length. Whether this will actually occur in a given case will depend more on how randomly the sequencing ...
Devon Ryan's user avatar
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4 votes
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checks for spike-in sequence controls

Disclaimer: I'm a developer for http://www.sequin.xyz. Sequin is a new set of spiked-in controls for next-generation sequencing, and that includes Illumina. We design controls for RNA-Seq, genome ...
SmallChess's user avatar
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4 votes
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What is the correct way to map Hi-C data with bwa mem?

See here, in particular slide #10 from the tutorial: bwa mem -5SPM ref.fa read1.fq read2.fq > out.sam Here, -SP disables ...
user172818's user avatar
  • 6,515
4 votes

Relationship between sequencing lane and ngs dataset

If you have used adapters containing barcode sequences (that identify each patient), you would presumably end up with one single tube with your libraries in it right? Correct, you have to prep each ...
Devon Ryan's user avatar
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4 votes

Tools for quality trimming at 5 prime?

You can have a look at cutadapt. It is capable of quality trimming for both ends as you can read here.
Mr_Z's user avatar
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4 votes
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What are some ways to error correct Oxford Nanopore long read sequencing?

How are you evaluating sequencing error rate? My most recent re-calls of 2017 sequences are demonstrating median single-read accuracy over 96%. Before considering Illumina, it'd be worth it to do an ...
gringer's user avatar
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4 votes
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What is the most accurate approach for de Novo sequencing?

HiFi reads can resolve most repeats in a genome. Illumina reads from these repetitive regions are likely to get mismapped and these mapping errors may become consensus errors. Most short-read ...
user172818's user avatar
  • 6,515
4 votes

How do I measure the proportion of different bacteria in a sample from a high-throughput sequencer?

If you've got a fast solid state drive with >200G of space, then kraken2 + bracken works really well on both long and short reads (e.g. Nanopore, Illumina). It's also specifically designed for ...
gringer's user avatar
  • 14.1k
4 votes
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How do I quantify a specific somatic variant?

I have a little function that can report this by using samtools to look at the read pileup. Um. It isn't the most elegant piece of code, but it does sorta do the job: ...
terdon's user avatar
  • 10.1k
3 votes

Relationship between sequencing lane and ngs dataset

It is worth noting that the Illumina NovaSeq and NextSeq address all of the lanes of the flow cell from a single loading point (by default). So on those instruments, you end up with all libraries ...
sjcockell's user avatar
  • 861
3 votes
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Why are there more barcodes than GEMs in 10X chromium data?

Your initial guess is almost certainly correct. I don't know about the linked read libraries, but in the 10X single cell sequencing protocol, separating real barcodes from noise barcodes is an ...
Ian Sudbery's user avatar
  • 3,311
3 votes
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GRCh38 centromeres mappability

should we expect Illumina 2x150bp (or shorter, 2x75bp) reads to map relatively equally to all centromere sequences? No. It has long been established that different chromosomes are associated with ...
user172818's user avatar
  • 6,515
3 votes

GRCh38 centromeres mappability

I doubt it, unless you are asking if mapping would be similarly bad for all centromeres. Here are some repetitive structures (probably not centromeric) that I've found in the nanopore reads for "human"...
gringer's user avatar
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3 votes
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Get gene names corresponding to CPG probes

Here's a Python script (e.g., called map_entrez_to_ensembl.py that uses mygene.info to convert a list of Entrez IDs to Ensembl gene names: ...
Alex Reynolds's user avatar
3 votes

Tools for quality trimming at 5 prime?

I never spent too much time on choosing my trimming software, therefore I might have missed some jewels. I use trimmomatic when I need versatility and I am entirely sure that it trims reads on both ...
Kamil S Jaron's user avatar
3 votes
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Singularity and Illumina's Nirvana

From your previous questions, it is apparent that your HPC has an odd singularity setup. You could ask you sysadmin about how to navigate it. EDIT After working with OP on chat (and based on previous ...
Ram RS's user avatar
  • 2,310
3 votes

paired-end short reads: will one file suffice?

I suspect you won't be able to avoid assembling or aligning. If you are working with strains that don't have their own, well studied reference genome and so want to avoid aligning so as not to bias ...
terdon's user avatar
  • 10.1k
2 votes
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Scaffolding a genome with hybrid data

Try LR_Gapcloser. I've used L_RNA_Scaffolder for trying to scaffold a genome (which turned out to be more complex than I had expected). It looks like LR_Gapcloser (written by the same people) is ...
gringer's user avatar
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