European Genome-Phenome Archive

File Quality

File InformationEGAF00002337383

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.

153 779 468262 512 378349 962 123393 750 056389 291 645347 112 877283 763 367215 891 423154 675 259105 265 28768 528 31542 985 75826 262 18215 732 2579 327 8325 551 7383 345 6832 080 7531 360 311941 303689 002533 897429 168360 363309 349272 386237 890212 011190 636172 373157 887143 192130 972118 792110 50199 15791 40383 13577 69671 13364 69859 32856 02451 85048 73945 37142 10139 71937 08335 20932 93731 76829 36527 57526 02624 60724 08422 85821 87121 16220 43920 10719 97618 64017 57517 15916 41315 93615 33314 73814 03914 02813 35813 35012 79412 62712 04411 80611 58411 16710 81911 08910 46010 78110 26510 43610 1219 4369 6219 2618 9308 4088 3598 2467 9767 8957 6147 6677 8387 7867 4967 3307 1637 0816 7016 9186 6576 5536 4466 3416 2156 2796 2876 1556 2086 2415 9365 7075 6095 7305 5175 8015 4675 3475 3575 4955 2965 1404 8824 8284 9824 9454 6254 8184 8594 6084 4964 4594 3424 3324 1103 9994 3263 8953 9303 9353 8573 8614 0033 9603 6023 8253 7223 6493 4903 5043 5063 5353 5623 3863 5423 5563 5443 4313 2073 3383 2333 1893 1713 0393 1822 9742 9362 8862 9272 8032 8582 8472 9082 8672 7732 6692 6622 6972 7812 7602 5802 7092 7012 6452 6782 6472 6102 5502 5582 4102 3712 4682 3402 2852 3702 4122 3362 3132 2522 2052 2962 2432 2552 1862 1862 0862 0272 0721 9851 9292 0602 1972 0672 2262 0982 1182 2002 1382 0571 9381 9711 8121 9501 9742 0712 0061 9661 9962 0492 0772 0721 9962 0592 0462 0342 1111 9602 0861 9551 9021 8501 9001 8491 8821 8631 8161 8931 8351 8461 8231 7831 7561 8061 8331 7341 6481 6631 6371 6441 6001 6431 5831 5461 5891 5091 5121 5421 6291 5161 5171 5861 5581 5521 6421 5151 6111 5571 4841 4181 4931 4671 4691 4621 5351 4541 4881 4741 4981 4821 4711 5271 4031 4001 4701 4831 4461 3641 2941 4191 4131 3311 3221 4331 4301 3931 3341 4291 3481 4521 3261 3291 2861 2751 2291 2341 2471 1901 1881 2451 2311 1621 2051 2851 2511 1881 1701 1771 2621 1661 1801 1361 1471 2231 2041 1731 2711 1371 1851 1101 1521 1901 1401 1451 0511 1761 1441 1701 1391 1021 1231 1311 0571 1621 1541 1351 1411 1551 2061 1341 1901 0651 0091 0631 0601 0131 0461 0189681 0339969761 013969936908919873950920968908958926934908911909888863871941884887912937994921880919891860871954808837868774769807775753780791763797773801761728742729771756754771727759762757763717727760760717648694667663660619629689630643670618624632608594656639647648631620626610651622609571487576640603537549550524649603617619624560575564543574573563602591580575585548601548551507518571563599594558599567570569543683558593610557538549551496516497528556579636568528520522487512490499481481450462545498559465500503500467483483507485524512511521502528436441452446463449446451438431454483446454448453504479453485516465473456461509440467451504419457429436452459432482451515469490439463450459475448464452464432478449468465464486488487439491455450416452456450451471470427436482456443416396408414454395410456416455402475399442379340362399381398389401409387409377368376399411360389426401413414361378393385370364347347324392366383383370354344313333349339358345358370362337371345318298329320334326337355385364356356361385378352373366310325315363316337342329350345373366368331367336366347350361329341303322325354346316337301365283297354339330309310285320304333297307321294282333266324321294297284284295266318287299264268269286314271286270267276241300273274267257268274273282264273274266255282288287289267282250272275279258252261307276275252259271254259253289250230260228243266264234239232233285265245263279256243273284254275271270260260225280257242275230235246264235256223215222229247217240241233235214216214213230251238244223231251223225220220204213229240243224197252230213256232232229210216205222199238191197197224216200214218221223183213217239199203211191197235221212209204190226187232176189201184197205197197182208183201189184185169193230202206212205213210204208182211192199208194210208214231182207210191189207177185216170210213202197192226196212183191190198172209176178190184186186173160174228 160100200300400500600700800900>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.

2 337 997000000049 392 226000754 132 915000000000430 826 4040000504 457 56000001 026 089 76000002 087 933 05700010 420 008 50300510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G# 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).

99.4 %100 567 95699.4 %0.6 %

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.

99.1 %100 286 50299.1 %0.9 %

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 %281 4540.3 %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 %50 580 06150 %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.

94.1 %95 197 28094.1 %5.9 %

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.

6.2 %6 296 6306.2 %93.8 %

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.

7 243 226139 099110 663173 055149 997166 573176 285232 181175 594187 776115 30990 92993 57394 00758 089101 85874 82285 346102 442143 804151 840167 809219 682146 905212 114299 75864 174486 69070 49959 45586 29699 63791 555113 66063 87667 86577 43596 62049 856146 3051 093 13791 43294 733143 828106 428169 680146 539215 736341 81076 81982 52479 84689 69460 284169 00982 11372 866151 80478 102121 44196 239 202051015202530354045505560Phred 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.

99.74%99.72%99.74%99.74%99.73%99.73%99.74%99.74%99.74%99.74%99.73%99.74%99.74%99.74%99.73%99.76%99.75%99.73%99.77%99.74%99.74%99.74%99.81%99.71%0.26%0.28%0.26%0.26%0.27%0.27%0.26%0.26%0.26%0.26%0.27%0.26%0.26%0.26%0.27%0.24%0.25%0.27%0.23%0.26%0.26%0.26%0.19%0.29%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped