European Genome-Phenome Archive

File Quality

File InformationEGAF00002485691

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.

363 402226 202124 101112 36388 21782 28973 74970 74363 35061 01056 91954 06752 26951 29448 16146 52643 61243 05640 07738 03736 69134 18232 82032 09931 08229 27727 73725 99025 67624 28322 19621 70820 72620 10218 54818 09617 07416 92016 20315 53214 72714 29013 23712 97612 07611 52710 93210 3789 5988 9808 6618 5797 5237 3597 0256 9466 1045 9515 3535 2744 9164 7974 2513 8953 7773 4993 5433 1942 6883 1292 7082 9242 4952 4082 1912 1271 9762 0071 7871 6981 8201 5681 5231 5071 4411 4121 3241 1871 2561 3901 1901 03996597982590184391872187672374469670358474563869048649049153240445536139739843132834032333030035126830534939826650436229626728926234027129434234220629322227622223723133019125017724017314315817022516917717423615016416813921518816614215514816412713313610114214796109103197187128127173951421039591105808783789591103102110148781537289108757583152156872221016975151135638714796986977647569741176566657076766265737062767173616612962544962576245127734962615676611346870707451137675854525268545056474648654868475311860535149604541464911949645936385746374245373435343838332943108374218452743303019403724372930422722352327342344402544324811129372431233490333932201002828303224252127261621152432232935282913202019152116291919191826232227183130283131323727303624352641232122343229222922213023241620232514241319432296132118161710111513221718651416172519231527162019222730202319212325272730221022181616141013131412151513162323161011191315212219171216151016121212121614121215814131415181591310181191518177961411131714914111682241313171616191518121212161921151717171510159131314121311171399141417822117181210181112101217121622181881892217131515111613211627252618141116816181414162315141722161320151821121622192129151921211120181319192516212225342822201519193721232324232527443023241425302626312525302822313335202426222948333329363031252824252124413041403127352933292127253328312927373340273433353326393324313931332833333723273739224030332630193132202324333234432724313227233530242199383226312331312431272724203228211899371921232719303729252421242616322835273627232428313527263627323925363231293129252730203522392326232226312910718342136322831202628232326332622182926212931283532303315182733222720241622291921182411221523221523132315131117261915121111141015219141718191922913811121527841924171918144209161613171989108715101016122411111917119131216199815999811141410714109149891610141471111914131712881317129121698102 693100200300400500600700800900>1000Coverage value10201002001k2k10k20k100k200k# 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.

10 47500000000000002 129 5670000000149 91900002 317 056000009 704 85700085 970 17600000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M# 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).

58.8 %786 54558.8 %41.2 %

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.

58.7 %785 52658.7 %41.3 %

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%).

0.1 %1 0190.1 %99.9 %

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.

50 %668 54750 %50 %

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.

50.1 %669 28850.1 %49.9 %

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.

6.3 %83 8596.3 %93.7 %

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.

729 3101 3052141 9229896231 2212 1123643 6718377502 0721 0923611 7023436157821 1332691 9291 5641 6822 6523 8473977 3404581 5067717542641 7802334985919973592 22125 4897835589421 6311 3611 9591 2141 3222 0951 6721 5907 9024701 7891 0933922 4071 330287520 295051015202530354045505560Phred quality score100k200k300k400k500k600k700k# 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.

99.83%99.84%99.87%99.85%99.78%99.9%99.87%99.8%99.88%99.84%99.91%99.92%99.85%99.91%99.89%99.88%99.87%99.89%99.89%99.87%99.88%99.65%97.73%99.96%0.17%0.16%0.13%0.15%0.22%0.1%0.13%0.2%0.12%0.16%0.09%0.08%0.15%0.09%0.11%0.12%0.13%0.11%0.11%0.13%0.12%0.35%2.27%0.04%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped