European Genome-Phenome Archive

File Quality

File InformationEGAF00005114896

File Data

Base Coverage Distribution

This chart represents the base coverage distribution along the reference file. Y-axis represents the number of times a position in the reference file is covered. The x-axis represents the range of the values for the coverage.

Data is represented in a log scale to minimise the variability. A high peak in the beginning (low coverage) and a curve descending is expected.

139 055 97535 735 4449 373 6242 493 953676 879203 21076 34740 36126 86119 55316 77215 27412 53911 4799 9309 3717 3887 4347 2566 6565 9925 2384 7024 7614 2174 0414 1833 8443 2733 2572 8843 0822 6572 5762 4922 4652 4382 3001 9881 7091 6182 1391 8301 8431 6261 5391 3491 3731 2901 4221 4601 2521 2021 0181 1571 1081 0601 0731 1541 1521 2661 0971 3211 1261 0071 03199779976794093980480083696786179495576670880177381572282073562572159663665370973958957359158148956650741235640539338239335133936225426827225720322431023827023520224130916419216119918319316616815515414913915615111112715620214113816716825615415817518511113414112615512787153959680841008987817986997988879392881019872835892681156810678101887813286906410887110704245585762946587604565485378696946585245524453396839373827433547385247302741252331263967253140284046354454535056708370283537505944513437291620371935204025222320272414282222243026254039361813143122253322272124292817393417222541192830403425343244423024191315191230384955172115211631132530181825152222202022221818199142525211728112172711191214131410137991211121761212162221211416101479151718141211117201051011471113912131415162513111822252612181414911101515141215161217231591312131912141811221114202741301211131925261430102717145111515131781419172920242316251515241820202118161312181917211710108101012141618131215131210121611121191571011101189165101399121278577888587108112124344597125735131415667563106210615465451115346555410446571353194321352312212551213654663248441121235612453456111585232212341212445314421111113121111111121121212111131111231122221333121255484136627816211132111131221113111211221111522564326121111121111111279100200300400500600700800900>1000Coverage value1101001k10k100k1M10M100M# Bases

Base Quality

The base quality distribution shows the Phred quality scores describing the probability that a nucleotide has been incorrectly assigned; e.g. an error in the sequencing. Specifically, Q=-log10(P), where Q is the Phred score and P is the probability the nucleotide is wrong. The larger the score, the more confident we are in the base call. Depending on the sequencing technology, we can expect to see different distributions, but we expect to see a distribution skewed towards larger (more confident) scores; typically around 40.

008 560 6490000000007 883 212000000000000010 618 8190000000261 147 80900000000510152025303540Phred quality score0M20M40M60M80M100M120M140M160M180M200M220M240M260M# Bases

Mapped Reads

Number of reads successfully mapped (singletons & both mates) to the reference genome in the sample. Genetic variation, in particular structural variants, ensure that every sequenced sample is genetically different from the reference genome it was aligned to. Small differences against the reference are accepted, but, for more significant variation, the read can fail to be placed. Therefore, it is not expected that the mapped reads rate will hit 100%, but it is supposed to be high (usually >90%). Calculations are made taking into account the proportion of mapped reads against the total number of reads (mapped/mapped+unmapped).

88 %2 664 36688 %12 %

Both Mates Mapped

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. This chart shows the fraction of reads in pairs where both of the mates successfully map to the reference genome. .

Notice that reads not mapped to the expected distance are also included as occurs with the proper pairs chart.

0 %00 %100 %

Singletons

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. If one mate in the pair successfully maps to the reference genome, but the other is unmapped, the mapped mate is a singleton. One way in which a singleton could occur would be if the sample has a large insertion compared with the reference genome; one mate can fall in sequence flanking the insertion and will be mapped, but the other falls in the inserted sequence and so cannot map to the reference genome. There are unlikely to many such structural variants in the sample, or sequencing errors that would cause a read not to be able to map. Consequently, the singleton rate is expected to be very low (<1%).

100 %2 664 366100 %0 %

Forward Strand

Fraction of reads mapped to the forward DNA strand. The general expectation is that the DNA library preparation step will generate DNA from the forward and reverse strands in equal amounts so after mapping the reads to the reference genome, approximately 50% of them will consequently map to the forward strand. Deviations from the 50%, may be due to problems with the library preparation step.

100 %3 027 122100 %0 %

Proper Pairs

A fragment consisting of two mates is called a proper pair if both mates map to the reference genome at the expected distance according to the reference genome. In particular, if the DNA library consists of fragments ~500 base pairs in length, and 100 base pair reads are sequenced from either end, the expectation would be that the two reads map to the reference genome separated by ~300 base pairs. If the sequenced sample contains large structural variants, e.g. a large insertion, where we expect the reads mapping with a large separation would be a signal for this variant, and the reads would not be considered as proper pairs. Based on the sequencing technology, there is also an expectation of the orientation of each read in the fragment.

The rate of proper pairs is expected to be well over 90%; even if the mapping rate itself is low as a result of bacterial contamination, for example.

0 %00 %100 %

Duplicates

PCR duplicates are two (or more) reads that originate from the same DNA fragment. When sequencing data is analyzed, it is assumed that each observation (i.e. each read) is independent; an assumption that fails in the presence of duplicate reads. Typically, algorithms look for reads that map to the same genomic coordinate, and whose mates also map to identical genomic coordinates. It is important to note that as the sequencing depth increases, more reads are sampled from the DNA library, and consequently it is increasingly likely that duplicate reads will be sampled. As a result, the true duplicate rate is not independent of the depth, and they should both be considered when looking at the duplicate rate. Additionally, as the sequencing depth in increases, it is also increasingly likely that reads will map to the same location and be marked as duplicates, even when they are not. As such, as the sequencing depth approaches and surpasses the read length, the duplicate rate starts to become less indicative of problems.

21 %635 52121 %79 %

Mapping Quality Distribution

The mapping quality distribution shows the Phred quality scores describing the probability that a read does not map to the location that it has been assigned to (specifically, Q=-log10(P), where Q is the Phred score and P is the probability the read is in the wrong location). So the larger the score, the higher the quality of the mapping. Some scores have a specific meaning, e.g. a score of 0 means that the read could map equally to multiple places in the reference genome. The majority of reads should be well mapped, and so we expect to see this distribution heavily skewed to a significant value (typically around 60). It is not unusual to see some scores around zero. Reads originating from repetitive elements in the genome will plausibly map to multiple locations.

558 0235 5446 2576 0766 0637 5806 9259 0259 3967 28119 1741 9793 2131 7611 5833 0201 3871 6441 8151 9114 8582 1772 4834 9563 91915 2348 3821 8783 7641 6941 1694 7251 7194 5491 9041 5543 9653 29015 3371 9001 1154 2351 7655 1612 0371 0935 1471 3343 2323 92083711 6401 3192 8593 0306015 5618995326 2142 225 879051015202530354045505560Phred quality score0.2M0.4M0.6M0.8M1M1.2M1.4M1.6M1.8M2M2.2M# Reads

Mapped vs Unmapped

Stacked column chart for both mapped and unmapped reads along all chromosomes in the reference file. It is a similar representation as shown in the Mapped reads chart but for each chromosome. Although sequenced sample may be a female, it is possible to get reads in the Y chromosome as there are common regions in both chromosomes called pseudoautosomal regions (PAR1, PAR2).

Unmapped reads belonging to each chromosome are determined when the one mate/pair is aligned and the other is not. The unmapped read should have chromosome and POS identical to its mate. It could also be due when aligning is performed with bwa as it concatenates all the reference sequences together, so if a read hangs off of one reference onto another, it will be given the right chromosome and position, but it also be classified as unmapped.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped