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 ...
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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'...
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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 ...
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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-...
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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 & ...
- 2,715
6
votes
Accepted
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 ...
- 504
5
votes
Accepted
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 ...
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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....
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5
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 ...
- 6,295
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, ...
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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 ...
- 13k
4
votes
Accepted
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 ...
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4
votes
Accepted
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 ...
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4
votes
Accepted
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 ...
- 6,295
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.
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4
votes
Accepted
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 ...
- 13k
4
votes
Accepted
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 ...
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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 ...
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3
votes
Accepted
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 ...
- 3,271
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 ...
- 5,467
3
votes
Accepted
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:
...
- 3,105
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 ...
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3
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 ...
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3
votes
Accepted
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 ...
- 6,295
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"...
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3
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). Each run also needs one MinION flow cell (\$900 USD).
...
- 13k
2
votes
Accepted
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 ...
- 13k
2
votes
Accepted
Viral Metagenomics
The specifics will depend on the experimental setup and data, but a few general comments...
1) Metagenomic data is often large volume and high in microbial diversity but low and uneven in coverage; ...
- 3,858
2
votes
How to align output of grep --color=always? (To QC fasta/fastq files)
I wrote a Perl script to do this. Just like zgrep, it can search through as many files as you give it. You can use the -c switch ...
- 9,352
2
votes
Genotyping with Illumina HumanOmni1-Quad before whole genome sequencing
The Illumina HumanOmni1-Quad beadchip is a microarray device consisting of 1,140,419 markers which have derived from the 1,000 genomes project. The markers chosen are is high-value regions of the ...
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