European Genome-Phenome Archive

File Quality

File InformationEGAF00002484481

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.

258 41776 78231 24723 07715 40713 21210 5599 7657 6937 3836 3515 6425 2444 7013 9124 2323 7663 9823 4713 1423 1773 0832 7872 6452 8632 3042 5302 2212 1762 3882 4352 3982 2452 1382 3032 4692 1311 9162 1101 9501 7961 8442 1421 8621 9211 7661 7531 9731 7291 5781 5701 7481 5611 7051 8071 6651 6161 4611 6421 6051 4541 3931 6041 3741 4531 2841 2691 3151 4831 2621 2601 2471 2971 2831 2941 2271 2521 1641 2521 2421 1881 2511 2781 2241 1721 2391 1951 2131 2331 1571 2351 1491 1101 1441 0641 0491 0831 0711 0289561 1081 0381 0619739799529949939861 0229581 0299969749799429979249691 0039839591 0329521 0899761 0059729729409758979699649269469299129891 041970895938907919848915833945858818835943916833787748868887832820848824883796785853925765758826797843815756798753804797788739793847765789753740793766798777767713688744805787765785795805766751772718658748679710691704745710748751728702704733685683726676689701666708681639617618639633586680650664639607603585655599626603617581568608564555560537570606564568547541521543512500494549496558519525519474497506484445518480486475504462456424429411414427449382389426430466445393390400345358371402345325374379357327344301329333335332342302355350353345367325352329321318321313328303317315292349297327314314296280275304269296271292284287253261255282272254239272238231212243202236207208206210241210226198193194204172175158159163176165193144162136169146147138136139143122130140129131135107106991018812310412911311791118104101103938389938210996968574811028372736367807155808072926563736659524471575154644853426544354653574539433540373545403340345040335223453746352937382547414834283637262930384233243329262924372722322022252821262230202526222422192214242117152923242128111918182935232419222618222919252526202231171928263323161622313224203122242329251536282021232129312228262824242523223026262217161919241920172627262627181823183425252530381529242726212534212322251819293430202827212522252525182122222722261626161418243118262528222329342624242920212223241628171218242125281822311423202423152818152129232725152016341815151819182013151313262526112920142118201920181417172315231523172020223317161911101719211572118221391021151515149191114112116171612781991212121212108111091319151211141391055913861314111781591691211914138101213171211101111141451111691181412587511579869656643111161055453781031269410796376313763410135722431323665410210782578215234243344312523112133314624121542242622524312232421311252142231112311231511233211412 906100200300400500600700800900>1000Coverage value1210201002001k2k10k20k100k200k# 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.

4 2800000000000000353 344269 1171 561 70300000000001 309 95863 7930503 009758 230184 356264 8501 737 4455 172 9032 005 9421 557 2543 687 11018 006 80345 645 65300510152025303540Phred quality score0M5M10M15M20M25M30M35M40M45M# 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).

73.3 %812 34073.3 %26.7 %

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.

69.8 %773 09269.8 %30.2 %

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%).

4.8 %39 2484.8 %95.2 %

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 %553 90550 %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.

60.8 %673 11060.8 %39.2 %

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.

13.8 %152 84013.8 %86.2 %

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.

350 7631 6091676364123105422871942 9677143 4941 4723804506 2934003 1711 6372193 116832 37813 997333 19034504480 3796838529662705 222125 33517052372408681 220801446 758962241824281462943325081 1422 616482 206051015202530354045505560Phred quality score50k100k150k200k250k300k350k400k450k# 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.

95.3%96.32%96.7%96.73%94.87%96.84%97.27%97.22%91.5%96.25%96.72%94.46%94.24%93.96%91.98%94.89%94.95%95.01%94.69%94.47%88.21%95.78%80.93%98.25%4.7%3.68%3.3%3.27%5.13%3.16%2.73%2.78%8.5%3.75%3.28%5.54%5.76%6.04%8.02%5.11%5.05%4.99%5.31%5.53%11.79%4.22%19.07%1.75%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped