Simon Andrews

Simon Andrews Posts written by Simon Andrews

I am the head of the Babraham Bioinformatics group. We provide commerical and academic bioinformatics services as well as manging the data pipeline for the Babraham sequencing service.

My group develops several software packages for processing high throughput sequence data including FastQC, BamQC, SeqMonk, Bismark and HiCUP.

Bioinformatics group, The Babraham Institute, Cambridge, UK

Illumina 2 colour chemistry can overcall high confidence G bases

With the introduction of the NextSeq system Illumina changed the way their image data was acquired so that instead of capturing 4 images per cycle they needed only 2. This speeds up image acquisition significantly but also introduces a problem where high quality calls for G bases can be made where there is actually no signal on the flowcell.

May 4, 2016 NextSeq, All Applications, Cutadapt, FastQC

Mixing sample types in a flowcell lane generates cross contamination artefacts

With the increasing capacity of a single flowcell lane it can be tempting to mix samples of different types within the same lane to make the most of your sequencing, but cross contamination between libraries in a flowcell can lead to the generation of artefacts which can mess up your analysis.

April 15, 2016 Illumina, All Applications, SeqMonk

Genomic sequence not in the genome assembly creates mapping artefacts

Probably the single biggest problem with the mapping of reads to a reference sequence is dealing with reads which come from parts of the genome which aren’t in the assembly. These reads can cause significant amounts of noise in anlayses performed on genomic data.

March 21, 2016 All Technologies, All Applications

MAPQ values are really useful but their implementation is a mess

One of the standard fields in the SAM/BAM file format is the mapping quality (MAPQ) value. This value can be very useful to help filter mapped reads before doing downstream analysis – unfortunately the implementation of this value is in no way consistent between different aligners so it takes a fair bit of research to know how to use it appropriately. Mis-applying the filter could cause reads to be inappropriately excluded from an analysis.

March 17, 2016 All Technologies, All Applications, BamQC, SeqMonk

Biased sequence composition can lead to poor quality data on Illumina sequencers

In some experimental designs a large proportion of the sequences in a library can have identical sequence at their 5′ end. These types of library can cause problems for the data collection and base calling on illumina sequencers, leading to the generation of poor quality data.

March 15, 2016 Illumina, All Applications, FastQC

Data can be corrupted upon extraction from SRA files

We are increasingly re-using data deposited in public sequence archives such as SRA, ENA or DDBJ and we rely on being able to successfully extract data from these sources. In some cases we have found that errors in the validation of the data can mean that data is corrupted when it is downloaded from these repositories.

March 3, 2016 All Technologies, All Applications

Barcode splitting doesn’t work as expected

When running multiple samples in the same sequencing reaction the different libraries are usually created with unique sequence tags (barcodes) to allow the sequence from the lane to be separated. Problems during this splitting are common and can have serious effects on downstream analysis.

February 12, 2016 All Technologies, All Applications, Data Processing

Deduplicating ambiguously mapped data makes it look like repeats are enriched

In a dataset where you have some degree of technical duplication, and have not filtered your data to only keep uniquely mapping reads then if you perform deduplication it will look as if repeat sequences are enriched.

February 11, 2016 ChIP-Seq, Data Processing, SeqMonk

Read-through adapters can appear at the ends of sequencing reads

Many sequencing platforms require the addition of specific adapter sequences to the end of the fragments to be sequenced. For an individual fragment, if the length of the sequencing read is longer than the fragment to be sequenced then the read will continue into the adapter sequence on the end. Unless it is removed this adapter sequence will cause problems for downstream mapping, assembly or other analysis.

February 7, 2016 Cutadapt, FastQC, Skewer, Trim Galore!

Libraries can contain technical duplication

The assumption when analysing sequence datasets is that every sequence comes from a different biological fragment in the original sample. Many library preparation techniques though include one or more PCR steps which introduce the possibility that the same original fragment can be observed multiple times, biasing the results produced. In some cases this type of duplication can be extreme and have a serious effect on the ability to analyse the data correctly.

February 6, 2016 FastQC, MultiQC, Preseq, SeqMonk

Mapping to a transcriptome can incorrectly report reads as mapping uniquely

For speed and efficiency many RNA-Seq mapping protocols map either entirely or initially to a transcriptome sequence. When the sample is contaminated with genomic material you can get significant numbers of reads reported as uniquely mapping when they actually map to many locations within the genome.

February 2, 2016 mRNA-Seq, SeqMonk

RNA-Seq samples can be contaminated with DNA

In theory RNA-Seq samples only contain reads derived from RNA. Most protocols include a DNase treatment to remove any remaining DNA from the sample. It is fairly common however to see samples with significant amounts of DNA contamination. Ignoring this can bias the counts generated from the data and throw off normalisation.

February 2, 2016 RNA-Seq, SeqMonk

Contamination with a different species you can guess

One of the biggest problems with sequencing libraries is that the material might be contaminated with something unexpected. One of the simplest forms of contamination is where you have material from a different species than expected. In many cases the rogue material will come from a species you can guess based on their other species commonly used in your lab. Screening for this type of contamination will help spot when you have contaminated samples, and can also help when you have completely switched samples.

February 1, 2016 FastQ Screen

Contamination with adapter dimers

The construction of sequencing libraries on many platforms requires the addition of specific adapter sequences to the ends of the fragments to be sequenced. Although there are steps in place to ensure that only valid adapter+insert combinations make it onto the sequencer it is possible to get adapter dimers with no valid insert making it through the sequencing process.

February 1, 2016 Illumina, FastQC

Positional sequence bias in random primed libraries

In a randomly primed library there is no reason to expect that a specific sequencing cycle should contain more of one specific base than any other cycle. It is commonly observed though that this type of library does contain a cycle-specific sequence bias, most frequently in the initial bases of the run.

January 31, 2016 mRNA-Seq, FastQC

Position specific failures of flowcells

Rather than a general loss of quality for a whole sequencing lane or run sometimes partial failures occur. These can affect specific regions and cycles and can have knock on effects for the data generated

January 31, 2016 Illumina, FastQC

Incorrect encoding of Phred scores

Base call qualities in fastq and BAM format files are recorded using a text based encoding. Mistakes in the way this encoding is applied can lead to incorrect interpretation of base call accuracies in downstream analyses.

January 20, 2016 FastQC, QC Software

Sudden loss of base call quality

Sometimes a sequencing run can experience a sudden and lasting loss of base call quality across all sequences.

January 20, 2016 Illumina, FastQC, QC Software

Loss of base call accuracy with increasing sequencing cycles

Illumina based sequencing shows a loss of base call quality as the number of sequencing cycles performed increases.

January 20, 2016 Illumina, FastQC, QC Software