European Genome-Phenome Archive

File Quality

File InformationEGAF00004839628

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.

91 127 671172 026 808257 024 097325 019 276360 845 366359 736 983327 198 101275 119 090216 077 213159 742 681112 110 13275 080 94748 392 95630 207 74818 403 83411 015 8336 565 6893 970 7292 462 4621 597 6911 109 175813 899636 103519 206438 688383 135335 708298 932268 102243 614221 132201 827186 617173 002161 528150 692141 459132 580124 982116 309109 003100 90993 50688 46582 25677 49373 07168 03464 13160 26756 74253 64450 38648 29345 42143 67841 10339 23237 10835 74435 70133 85031 64730 70429 53327 99026 95425 61425 32124 87024 09923 55623 02022 11021 93721 21120 30120 03119 50219 16718 91318 35717 98017 89616 93116 65616 47716 14715 43015 15114 35814 13514 23513 77213 00513 09813 06512 42012 16912 13511 83211 72011 39711 23810 72810 64510 59310 24210 0509 7239 7529 7059 3418 8879 1059 1228 6218 7098 5838 4658 2598 3618 0837 9417 8687 8027 6847 5007 4907 4137 2816 9636 9517 0467 0616 4706 7056 6596 8306 5146 5426 4396 7056 5056 4596 3556 3096 0736 1525 9646 0435 8145 7295 6365 5095 6195 6515 3825 4615 2755 3565 3195 3865 2755 1144 9524 9464 9185 0215 0214 9775 0094 7784 6044 5734 6144 5764 5464 3634 3994 3094 2134 1504 0394 0094 0804 0934 0614 0273 9083 8414 0173 8343 7393 6923 6743 5823 7513 7413 6553 6883 5573 6563 6463 7163 4683 4263 4843 5203 2973 2553 1593 2323 1413 0693 1373 0432 9792 8943 1222 8952 9132 8942 8212 7012 8272 6662 7362 8172 6312 6682 7162 7612 7572 6892 6752 5942 5152 5372 5552 4352 4632 4302 3542 4292 3952 4032 3932 3812 3092 2572 3862 1292 2852 1552 2882 2022 2092 2492 2412 2802 2182 1972 1912 1502 0402 0122 0952 1212 1131 9892 0311 9291 9581 9901 9692 0132 0282 1292 0872 0422 0051 9941 9601 9611 8651 7611 8851 8271 8231 8561 8351 8801 7671 7671 7821 7051 6891 7491 7221 8251 7471 7581 6571 7551 6881 8261 7551 7681 6541 6131 5201 5411 5901 5761 5741 5881 5481 5011 5421 4601 5031 5091 4751 5471 4981 5021 4921 4661 4521 4771 3651 4441 3931 3771 4091 4111 4771 3851 4361 3821 3281 3791 3461 2801 2921 3511 2651 3091 3461 3521 3111 3551 2441 2451 2841 3501 3091 2991 2051 2071 2541 2301 2301 1721 1151 2191 1711 1571 1261 1771 1291 1681 1661 1891 1351 0361 1291 1281 1761 1561 1211 1311 1181 2381 1411 2121 1081 1621 1861 1101 1311 1141 0991 0591 0791 1221 1101 1421 0711 1151 0841 0971 0359551 0001 0111 0211 0021 0381 0181 0239519681 0599399299609799969091 0009579179539839819861 0149888818859148788909029159309378989259279229168559088498989269159559399539359309641 049941895941857934936834920847854882843854878838836805809749770825833807791789867835758789792825775805789763820740802772786796784772759775712731776753723747709655717705754682696713680711747707732733670703730698671666652662660662724674630658635683650679687667651636637609613638620622642646657658680685633676663659624582628588598584658640640592659640627608611619641677646609594585603569552612582575602532569538529553602582543600606578578587513537518553557546528500540523537536538493550520535504494524447500510518490557565516514504574529544523539528572548573554541515510481562517516551518541525519510550508531521524548496553477468534502507540491509572480479457445519476465484475504467478471485482519466482459469434422464444476421438453492444461424399442460463452389436479390414414442427420470446417452446439439427412408449425408407403358390372343402406393332355323371372366394387402395439376444431446398372404379420366391369368382407391413419387403404447401415443409425423438406435423402427402436419409397415375409420418422417414398384403369420390404435337413418417392383412370378410371390401359373395412431385398377400372392411407395422418389379385372408364357352385368357379349377406367370370374335365405402375396360359391376413409418357360360385365373368378358365378367359358357350338352328362345344353344353329329299295337338335323320330341311319306301285307296308306310343323315327339317303311309282304288294277284305313296311314331306312344312278297306280238286289317291313295253309263261286287285287270279297279299292279304262297283338296295305303289267275286337360261262242274272265258257266244251254261291248237237280238239260253 100100200300400500600700800900>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.

1 600 296000000010 802 650000774 738 125000000000448 119 2940000524 516 48600001 155 242 45800002 561 860 44900014 090 916 09400510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G10G11G12G13G14G# 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).

99.7 %129 224 47399.7 %0.3 %

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.

99.6 %129 026 81899.6 %0.4 %

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 %197 6550.2 %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 %64 794 02650 %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.

97.3 %126 069 52297.3 %2.7 %

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.

7 %9 064 2837 %93 %

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.

6 284 472128 48978 602154 188113 026121 229135 920184 14988 927143 89366 57057 73376 91889 65747 560108 06976 67786 500109 066154 445156 222155 166190 719148 127233 637381 95127 512649 53739 15836 69367 90275 46136 84891 63337 30437 23157 91578 62122 705118 4191 573 06381 11179 366134 224113 669209 853179 926277 158433 67653 68566 63561 05475 20036 73677 93468 53149 727174 94053 102104 621116 713 526051015202530354045505560Phred quality score10M20M30M40M50M60M70M80M90M100M110M# 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.

99.84%99.84%99.84%99.84%99.84%99.84%99.84%99.84%99.84%99.84%99.84%99.85%99.84%99.85%99.84%99.86%99.85%99.84%99.87%99.84%99.84%99.84%99.9%99.85%0.16%0.16%0.16%0.16%0.16%0.16%0.16%0.16%0.16%0.16%0.16%0.15%0.16%0.15%0.16%0.14%0.15%0.16%0.13%0.16%0.16%0.16%0.1%0.15%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped