European Genome-Phenome Archive

File Quality

File InformationEGAF00002307084

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.

187 202 976303 678 733385 315 580413 117 930388 881 194330 487 048258 414 894188 832 169130 566 51186 360 93054 997 35134 054 00320 665 51812 435 9027 464 1924 525 6692 805 0111 787 0121 190 832840 303626 537494 107399 773332 927282 323244 708214 040190 987169 294153 641139 047127 269115 501104 24097 08788 86179 53274 69969 61563 41157 22554 09950 65847 27243 92341 07439 43437 48135 30333 20431 69429 65028 47727 13925 61225 25323 92022 85921 68120 74320 23819 22119 24318 59117 75716 81316 23315 54815 54414 80614 68414 37713 55313 20313 32012 82212 26912 11312 09511 73211 93811 05610 78010 23510 0659 9039 4859 3319 0519 0908 8698 6298 5618 4668 6808 2337 9327 7737 8457 5407 1007 2037 0156 9736 9186 7066 4236 1695 9575 7155 7135 6195 4275 5095 7145 6375 1685 1225 0585 0265 0945 0594 8734 8104 4814 4394 3114 2574 3454 2834 4734 1834 1764 1954 0573 9993 8613 7753 7203 5623 4793 5123 4383 2543 3793 3213 4083 1853 3273 1923 0513 0522 8852 8762 9112 8742 8882 7462 7252 7242 7172 7202 5452 5682 5192 4992 6582 5662 4172 4432 2892 3832 3402 4282 3732 3342 1972 2402 2882 1252 0382 1672 0542 1202 0832 0872 0422 0842 0102 1542 0391 9281 9241 8611 9731 9381 9541 8001 7561 9541 8311 8131 8491 8631 8641 8121 7411 7461 6891 6481 7491 7601 7631 7701 6621 7261 6641 6461 6331 5951 5371 5851 6141 5511 5271 4971 4721 5801 5321 5721 5911 5501 5621 4931 4531 4601 4281 3901 3611 3221 4041 4041 3791 3731 3401 2751 3691 3431 3361 3321 2711 2741 2061 2541 3001 2811 3071 2421 2701 2511 2811 2741 2871 3251 2441 3541 2691 2321 1561 1091 1151 1881 1641 1131 1621 0571 0411 1301 0501 0741 0361 0331 0711 0071 0941 0691 0611 0901 1171 0271 0151 0409979801 0661 0011 0189989519681 0329991 0149329409201 0101 1141 0441 0471 0039901 0111 020934947919991917869859851781832829807900840878819862777859818863794838851792851780809800790867810789813820725772728778740749769778748685776658746772707711703635691755676699680676694681716752660686669653727726673699773728715699745786717735756791774737714713757710700637697736667677712658665695666674685678694683684680674697648641671648689650671686732662683650673689611652621623657638607628658631660666715659685663673641635653694693676674677692692664691706734776730736701687748716727772726693692744659712689650668675700684691689665754674606622598586658611641640615587572629591570565682594571582541537542541599522571555528578540574553579570545576590633530539605531577568561563526579543530530524528476522469505471498479485485453461452418396385424452404393437409396387389393379348382356355332320331344348348327328353359374360349293340358337322342338371322315282286298290297292306308313279307293297296290281272294296280271284269265293281261274266279267242256263256243252276223274263263227254246243232229234262250257287292256243208208244230243264243208254253282265263263274263257224220227235244234239250207229219199223229217254245250245238225258246248222233233237252246273194285261239220224213231233233216225213237218226206219262232220202195223229200215212216211222174214215218220236215251205235232207225248232249213210191192209211230204202203230221229227215186195199200190193204208222213232226222200195187202196235177199173198218176221178180203172195196204191177178195193211195214204218188199204208197218225173200187203191214196201193190203181187205202213191194195208202218208231232198227179218214219206230232240247253255254271240245249229247238275269249237245248263255247253276283241243286263264282266232280246248258264279276228268251247242245227271244255276246241261247242279268236261259257271243249262250249237246228273228235248256255262216253236236247232250234255225248249213226256218226230230238198249205241241206223222225217223184183200213203210201199199205234216205203186226254199194206223204206182216216232208175208207225214212202188195193198210199197200182130 314100200300400500600700800900>1000Coverage value1k10k100k1M10M100M# 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.

1 923 6120000000117 527 832000799 135 992000000000487 331 6570000600 457 86500001 075 698 57300002 093 698 6690009 986 058 48800510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G# 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).

99.5 %99 910 39399.5 %0.5 %

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.

99.3 %99 710 91099.3 %0.7 %

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%).

0.2 %199 4830.2 %99.8 %

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 %50 204 74450 %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.

96.3 %96 715 89696.3 %3.7 %

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.

11.7 %11 791 14411.7 %88.3 %

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.

4 406 85874 93950 33193 06071 00976 84483 390111 61562 57986 84342 15234 94745 28049 22226 71560 53642 71946 41555 63773 87673 28685 607106 69981 932132 893223 93919 058434 13323 69321 82142 06943 94326 59554 21123 70823 54835 66645 40815 96473 204988 72554 29653 74789 29674 067135 506120 980188 010305 51638 98349 03444 01755 27629 14961 20450 37538 782127 18941 07077 19492 365 654051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M# 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.

99.8%99.78%99.8%99.8%99.8%99.8%99.8%99.8%99.8%99.8%99.8%99.8%99.79%99.8%99.8%99.82%99.82%99.79%99.83%99.81%99.8%99.8%99.85%99.75%0.2%0.22%0.2%0.2%0.2%0.2%0.2%0.2%0.2%0.2%0.2%0.2%0.21%0.2%0.2%0.18%0.18%0.21%0.17%0.19%0.2%0.2%0.15%0.25%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped