European Genome-Phenome Archive

File Quality

File InformationEGAF00002485410

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.

893 960247 947138 799107 77084 19875 39166 66060 88356 12552 95248 89047 52644 44842 67739 63538 43038 37037 80235 92035 28534 22035 08233 65834 07532 88533 05731 99231 17730 12529 77728 87929 68928 44828 25127 78126 81326 64125 53425 13524 91424 57623 86123 38222 16221 35720 78520 38120 07719 64218 59418 59718 58117 45617 25216 74015 82515 29115 16414 82414 15613 79313 69012 94412 52312 17511 79511 40411 20111 00110 75010 20910 7029 9349 7829 2298 9278 7288 7828 4518 2247 9007 4037 2667 2477 0816 6766 5016 0826 1435 7675 6095 6525 3515 2705 1234 9824 7354 6654 3124 2104 2033 8583 7643 9223 7073 8253 4823 4423 3663 0312 9443 0183 0272 7832 6562 5832 5602 4562 4932 3712 2972 2682 1672 0172 0022 0802 0141 8801 8901 7991 7531 8361 5531 6181 5251 5321 5071 5551 4071 3441 2811 3751 2961 2231 0911 0111 0409831 0719779238518818128188928418107586747167167316956336906166276445605825315375295065225024694444495055394754184354664813723573853463974023303593363603363843163132853033003052542732352392552482612592382462542022352312432162222172292232022261971951861911821861881581741421631861941691691751532021801612021331451581561431491412011241561571279112511811413910212195941151801201281031221091121391091061201041001011201101161161179274841029891849393958882555664628677809792548155858361728566807669477880967291667360696674808579677367796362687664686970627573631047780736159716674726856676063647768677570495666507177747359656553665475735955681235454466849495467436162486273596856445570546053735557535750525066676159425144435040545254545672493834454628323555354354413755445548385359504649456259564745456351465053565139463755544351577552374246475252425146483840384943474253404447294144515562524239303640493838333335273437323829343640353441253726364130384241224333393837333450364142355836313740473645423430403733353830382539443440293532333430284538454135363337324841302647373338334337333628364036312638663437413232384343393428382235332629284128212931262835382435182417312818202526191514211920201517252118192717191820262720112323211424232419152617201819262122201715172619242218192616151821182124172017202024231918182013261818121313131614182314131421201412131829192516161421262426161713183015162213142313101715161224202314132311161414141555131610112010151011101916171111611175916121617131012714149161413914910168118111081796131013111010128964148891081051013667877638569631134668066388479556636341063132424124152272231111221215523411135111111421211643324221211212311611124231 860100200300400500600700800900>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.

2 32100000000000005 290 6180000000294 27700005 174 6660000011 141 17100087 180 24700000510152025303540Phred quality score0M10M20M30M40M50M60M70M80M# 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).

98.7 %1 435 64198.7 %1.3 %

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.

98.6 %1 433 60098.6 %1.4 %

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%).

0.1 %2 0410.1 %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 %727 22250 %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.

79.6 %1 157 17679.6 %20.4 %

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.

10.2 %147 95410.2 %89.8 %

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.

167 4011 8985453 5068838982 0072 6036994 6631 6011 4645 3301 9601 0203 1055831 0041 5691 7654123 1521 5242 2253 3445 88094511 0451 1094 1211 8641 5768403 6015401 1981 2951 8738774 42936 3011 8861 3331 8744 3622 1931 6262 2652 3014 1973 5083 52314 6709323 4232 6227834 8742 8585891 134 515051015202530354045505560Phred quality score0.1M0.2M0.3M0.4M0.5M0.6M0.7M0.8M0.9M1M1.1M# 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.

99.85%99.88%99.86%99.9%99.84%99.83%99.85%99.83%99.87%99.9%99.9%99.85%99.86%99.88%99.84%99.83%99.83%99.89%99.77%99.86%99.91%99.83%99.55%99.92%0.15%0.12%0.14%0.1%0.16%0.17%0.15%0.17%0.13%0.1%0.1%0.15%0.14%0.12%0.16%0.17%0.17%0.11%0.23%0.14%0.09%0.17%0.45%0.08%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped