European Genome-Phenome Archive

File Quality

File InformationEGAF00002485472

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.

878 407410 765239 195199 775160 217146 111128 297121 317112 273106 522100 58298 64397 15794 37989 86488 27287 06184 19481 79478 98976 77175 61874 62873 31171 20369 77368 36465 79564 78662 57961 32460 08957 16055 60253 68052 85250 92149 08646 69245 14443 01741 93040 08738 68936 11334 81134 53233 36031 02330 22629 05327 77426 52226 08724 64423 56922 68321 33420 56919 70718 65817 85316 78416 28715 49914 53813 99913 05712 74511 79611 56010 78610 2069 8489 3508 7138 7788 3247 8927 4386 8056 4906 3505 8905 6715 1294 9034 6224 3794 3423 9893 8433 8713 6843 5113 2483 1702 9403 0762 5582 6282 4912 3092 3522 0742 0932 0461 8461 7781 7081 6211 7461 7411 6361 4481 5171 3971 2951 3281 2401 1751 0911 1511 0541 0021 0631 00087591688878373378273166376764670664759558055055460357456854953250752845455546047548039643441647749146743641840139634438841232536443832934332231836433027531529829530337331526128629526922422226925131025222422524126723220628921631129523425223927127821724125121020420918218817915920118123919219818420420917918116216817318223118717016817817616219618315519415715512515216216316117515716313914815512312914617320313112611010612299174114106139128123118119144117122130120136111130119125115939913510096179103889597769410111410082978499857981679584951248091919110166738594987374856883867595215120887573769158121728878665063666976627264103725555566661456065485374787476605949715442505056423851495866574860585562635051555148745067613846645958525959686751474250514741555548444954504753533035435733324429455536464435475439445548402938303128303143404329413039443339333532343444444026253440293626372932352529232924323230322427183232383528302928274034314942323936383333283523322123322924343326193030202034354324182125292923191923231624261921222816192416203024212520232218161715252013262618163327252030282422283531192432202236303535233126193427302774392722192130242817212223383119201833352028254117253127162224172822211729232317222216121415161915201922261518182215322224232722202416271617191726292622251724253122282725312522231722292929242424273034312017262220262031222138222518244135163532363424353138292523354228422941382926334235334429343629433839353536374435374238353256394725353334362730403022413242393134193823263234222435382536342328212129432339273322242821213130292729334025262544272730272242242328223117172927201829231523262815151721221723181530232518232925283222282225172424222831321719181828213130312929233219192340333939332926413836203122323523242426203015232129214126222417292328302322262433172529302420232318161814152134212017201119201719181817211415241827201826231717152719151819131522171118181313101714181317172123201625156 022100200300400500600700800900>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.

173 59300000000000005 523 7620000000285 87400005 631 6790000015 176 247000139 603 69500000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M90M100M110M120M130M# 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).

88.7 %1 967 11988.7 %11.3 %

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.

88.4 %1 961 60288.4 %11.6 %

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%).

0.3 %5 5170.3 %99.7 %

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 %1 109 29950 %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.

75 %1 664 40275 %25 %

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.

8.8 %194 7208.8 %91.2 %

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.

469 1591 5565253 2051 0648381 7291 9848173 8121 4221 3894 8201 5589032 6217139071 2761 3075822 7981 6462 1513 0894 31887417 0449703 8801 8251 3299323 8005899661 3241 2978564 34942 8801 6631 4022 0004 1852 5512 1792 5112 5984 6033 8694 14220 1221 0034 6112 3547928 2312 6525851 586 751051015202530354045505560Phred quality score0.2M0.4M0.6M0.8M1M1.2M1.4M# 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.

99.68%99.75%99.77%99.63%99.65%99.76%99.78%99.68%99.62%99.73%99.82%99.8%99.63%99.82%99.72%99.61%99.71%99.79%99.77%99.75%99.7%99.41%93.74%99.9%0.32%0.25%0.23%0.37%0.35%0.24%0.22%0.32%0.38%0.27%0.18%0.2%0.37%0.18%0.28%0.39%0.29%0.21%0.23%0.25%0.3%0.59%6.26%0.1%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped