European Genome-Phenome Archive

File Quality

File InformationEGAF00002445885

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.

784 866168 51760 73439 11326 42620 81615 39012 42510 2428 9138 3528 5007 6436 2086 4006 1195 6695 3205 0825 0984 9894 6754 7444 0184 4564 4183 8984 3503 9943 8914 3474 1553 6963 9803 8403 7593 5943 4973 5963 4553 6523 4673 4593 3143 3583 2213 0793 0453 1303 0472 9913 2993 2982 9523 2013 1273 0792 9863 0093 2183 0953 4373 2543 1033 0582 9402 9313 0273 0703 1383 0403 0252 8733 0803 1813 2022 7992 9782 9012 8862 7982 8922 9862 8822 8792 8862 8462 7572 8812 8032 8202 9132 8392 7042 8062 8862 8722 5532 6382 6442 6862 7072 8152 6522 7582 8352 8812 6232 7322 7902 6042 6462 7332 8082 5372 6552 7592 6282 4572 5512 6232 6302 5652 5402 7882 5452 6562 5972 5932 6302 6532 5742 5292 5832 5412 6612 5782 6222 5332 5352 5492 4302 5992 5732 3622 3792 5012 3722 5162 3502 4852 3492 3662 3432 5542 4072 2802 3532 2352 3132 2442 3932 2402 4092 2332 2042 2852 2832 1392 1172 1832 1611 9281 9722 0152 0642 0221 9841 8921 9801 8561 8541 9921 9011 7781 8251 8451 8151 6921 6781 7631 7651 8061 8371 7311 7221 6811 6211 5861 6171 5641 6071 5431 5481 5271 5401 4611 5771 4921 5491 5171 4341 5511 5361 4861 5291 4201 3081 3161 3441 4021 3011 2181 2491 2481 1731 1701 1391 2181 1301 1511 1321 1401 1311 0599241 0709941 0281 1041 01099394689791693496286091889489088088086281282778484077981673871975970074166462368867664460966659766661855557355757658555751954854358752952848248744744453147946246755050942644942039339338540136237540331733937838537337130532534433928732635229428328030421125822524627123326120628718720925918521618521619622820817717417713815515015213916914013613111212112714012710814512011414510610796851168011194109938889958279837885768968718110085698183707056755671707368395060726444535147514544524449624947433238414532283830374538322428244131583731453540434228272431373941273235272725424437332123354030261636352934303531382415282928202123222819212321222511202314141724203616202321171317242218151821141416142119192015261414201619152419201416151416172713211313171018241319161515182416241333153017271819157211919251219181412271526181422127181720182416181820211722202124192315241815202217146141461323131516131914101213141582112222519151716131811151014171412117191512922767121219141912139155109715101320101518111111710713168561681315613161312122151010257108101110681165955879916121687911710121146912111111711467441088101461071413897986148111181110117310667911479827628482683369674372532541012873121085634635132241010462545216658511591037557752843895436456924455664747365737656271226226774657578347375106445423211334572245363662448324295397669883683484762342143324315223322663734233531213432 304100200300400500600700800900>1000Coverage value1101001k10k100k# 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.

7 36400000000000003 309 0220000000158 77200003 378 149000008 536 41700076 799 82600000510152025303540Phred quality score0M10M20M30M40M50M60M70M# 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).

98.3 %1 208 68198.3 %1.7 %

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.

98.2 %1 207 46098.2 %1.8 %

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%).

0.1 %1 2210.1 %99.9 %

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 %614 59750 %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.

82.4 %1 012 75482.4 %17.6 %

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.

12 %148 01712 %88 %

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.

98 2811 1864662 1705485151 0081 1746492 6728511 0203 6019546952 0643798219047822721 9427761 4692 1912 94176812 3217872 7391 1719546172 3812856437209206082 66728 4401 2278201 3042 6481 7061 4111 8971 6223 1762 6723 56814 0336982 9111 7277025 2391 6903951 016 766051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M# 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%99.93%99.91%99.92%99.9%99.91%99.93%99.93%99.88%99.92%99.93%99.95%99.91%99.94%99.89%99.87%99.89%99.89%99.92%99.94%99.76%99.75%97.92%99.97%0.16%0.07%0.09%0.08%0.1%0.09%0.07%0.07%0.12%0.08%0.07%0.05%0.09%0.06%0.11%0.13%0.11%0.11%0.08%0.06%0.24%0.25%2.08%0.03%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped