Adapt Pt Tutorial
Boyan Atanaschev
Are you just using the default parameters or have you used the parameters I used? In the ZIP file I attached yesterday, you should be able to see a properties.xml file that details all the settings I used.
I will update the documentation when I get a chance and put together a tutorial. In the meantime, are you free tomorrow? I will be taking part in the imagesc-live event and could walk you through adapt then? More details here.
Also, one other question: so I sent you two files, one is GFP.tif and the other is RFPmem.tif. The GFP is supposed to be the cytosolic signal, and the RFPmem is the membrane. At any point during the analysis, did you have to use both files? Or did you only use the GFP.tif to generate those outline contour images? I was under the impression that ADAPT needs both files to work or am I misunderstanding again?
I will update the documentation when I get a chance and put together a tutorial. In the meantime, are you free tomorrow? I will be taking part in the imagesc-live event and could walk you through adapt then?
Also, one other question: so I sent you two files, one is GFP.tif and the other is RFPmem.tif. The GFP is supposed to be the cytosolic signal, and the RFPmem is the membrane. At any point during the analysis, did you have to use both files?
In the preceding tutorials you created a custom business object with a simple data structure and its persistence. Afterwards you generated an UI for this business object and exposed it as a Fiori Launchpad application. As the generated User Interfaces only lists all fields of a business object node, adapting the UI might be necessary to improve usability of it.
The sequence of the adapter is given with the -a option. You need to replaceAACCGGTT with the correct adapter sequence. Reads are read from the inputfile input.fastq and are written to the output file output.fastq.
Cutadapt searches for the adapter in all reads and removes it when it finds it.Unless you use a filtering option, all reads that were present in the input filewill also be present in the output file, some of them trimmed, some of them not.Even reads that were trimmed to a length of zero are output. All of this can bechanged with command-line options, explained further down.
Supported input formats are FASTA, FASTQ and unaligned BAM(uBAM, only for single-end data at the moment).Supported output formats are FASTA and FASTQ. Compressionis supported in multiple formats and detected automatically.
The output file format is also recognized from the file name extension. If theextensions was not recognized or when Cutadapt writes to standard output, thesame format as the input is used for the output.
When writing a FASTQ file, a second header (the text after the + on thethird line of a record) that possibly exists in the input is removed.When writing a FASTA file, line breaks within the sequence are removed.
Cutadapt supports compressed input and output files. Whether an input fileneeds to be decompressed or an output file needs to be compressed is detectedautomatically by inspecting the file name: For example, if it ends in .gz,then gzip compression is assumed
Cutadapt supports parallel processing, that is, it can use multiple CPU cores.Multi-core is not enabled by default. To enable it, use the option -j N(or the spelled-out version --cores=N), where N is thenumber of cores to use.
To automatically detect the number of available cores, use -j 0(or --cores=0). The detection takes into account resource restrictionsthat may be in place. For example, if running Cutadapt as a batch job on acluster system, the actual number of cores assigned to the job will be used.(This works if the cluster systems uses the cpuset(1) mechanism to imposethe resource limitation.)
Make also sure that you have pigz (parallel gzip) installed if you usemultiple cores and write to a .gz output file. Otherwise, compression ofthe output will be done in a single thread and therefore be a bottleneck.
Option -Z (equivalent to --compression-level=1) can be used to limit theamount of CPU time which is spent on the compression of output files.Alternatively, choosing filenames not ending with .gz, .bz2 or .xzwill make sure no CPU time is spent on compression at all. On systemswith slow I/O, it can actually be faster to set a higher compression-levelthan 1.
Cutadapt can do a lot more in addition to removing adapters. There are variouscommand-line options that make it possible to modify and filter reads and toredirect them to various output files. Each read is processed in the followingorder:
The requirement for a full match at the beginning of the read is relaxedwhen Cutadapt searches error-tolerantly, as it does by default. Inparticular, insertions and deletions may allow reads such as these to betrimmed, assuming the maximum error rate is sufficiently high:
The B in the beginning is seen as an insertion, and the missing Ras a deletion. If you also want to prevent this from happening, use theoption --no-indels, which disallows insertions and deletions entirely.
If you anchor an adapter, it will also become marked as being required. If arequired adapter cannot be found, the read will not be trimmed at all even ifthe other adapter occurs. If an adapter is not required, it is optional.
As described, when you specify a linked adapter with -a, the adapters that are anchoredbecome required, and the non-anchored adapters become optional. To change this, you caninstead use -g to specify a linked adapter. In that case, both adapters are required(even if they are not anchored). This type of linked adapter type is especially suited fortrimming CRISPR screening reads. For example:
All searches for adapter sequences are error tolerant. Allowed errors aremismatches, insertions and deletions. For example, if you search for theadapter sequence ADAPTER and the error tolerance is set appropriately(as explained below), then also ADABTER will be found (with 1 mismatch),as well as ADAPTR (with 1 deletion), and also ADAPPTER (with 1insertion). If insertions and deletions are disabled with --no-indels,then mismatches are the only type of errors.
The level of error tolerance is determined by a maximum error rate, which is0.1 (=10%) by default. An adapter occurrence is only found if the actualerror rate of the match does not exceed the maximum error rate. The actualerror rate is computed as the number of errors in the matchdivided by the length of the matching part of the adapter.
Relating the number of errros to the length of the matching part of theadapter is important because Cutadapt allows for partial adapteroccurrences (for the non-anchored adapter types). If only the absolutenumber of errors were used, shorter matches would be favored unfairly. Forexample, assume an adapter has 30 bases and we allow three errors over thatlength. If we allowed these three errors even for a partial occurrences of,for example, four bases, we can immediately see that this results inunexpected matches. Using the error rate as a criterion helps to keepsensitivity and specificity roughly the same over the possible lengths ofthe matches.
The -e option on the command line allows you to change the maximum error rate.If the value is between 0 and 1 (but not 1 exactly), then this sets the maximumerror rate directly for all specified adapters. The default is -e 0.1. Youcan also use the adapter-specific parameter max_error_rate or max_errorsor just e to override the default for a single adapter only.Examples: -a "ADAPTER;max_error_rate=0.15", -a "ADAPTER;e=0.15"(the quotation marks are necessary).
Alternatively, you can also specify a value of 1 or greater as the number ofallowed errors, which is then converted to a maximum error rate for each adapterindividually. For example, with an adapter of length 10, using -e 2 willset the maximum error rate to 0.2 for an adapter of length 10.
The value does not have to be an integer, and if you use an adapter typethat allows partial matches, you may want to add 0.5 to the desired number oferrors, which achieves that even slightly shorter than full-lengthsmatches will be allowed at the specified number of errors. In short, if youwant to allow two errors, use -e 2.5.
This also works in the adapter-specific parameters.Examples: -a "ADAPTER;e=1", -a "ADAPTER;max_errors=2.5". Note thate, max_error_rate and max_errors are all equivalent and thedecision whether a rate or an absolute number is meant is based onwhether the given value is less than 1 or not.
Any N wildcard characters in the adapter sequence are skipped whencomputing the error rate. That is, they do not contribute to the length ofa match. For example, the adapter sequence ACGTACNNNNNNNNGTACGT has a lengthof 20, but only 12 non-N-characters. At a maximum error rate of 0.1, onlyone error is allowed if this sequence is found in full in a read because120.1=1.2, which is 1 when rounded down.
This is done because N bases cannot contribute to the number of errors.In previous versions, N wildcard characters did contribute to the matchlength, but this artificially inflates the number of allowed errors. For example,an adapter like N18CC (18 N wildcards followed by CC) wouldeffectively match anywhere because the default error rate of 0.1 would allow fortwo errors, but there are only two non-N bases in the particular adapter.
Since Cutadapt allows partial matches between the read and the adapter sequencefor most adapter types, short matches can occur by chance, leading to erroneouslytrimmed bases. Forexample, just by chance, we expect that roughly 25% of all reads end with a basethat is identical to the first base of the adapter. To reduce the number offalsely trimmed bases,the alignment algorithm requires that at least three bases of the adapterare aligned to the read.
This minimum overlap length can be changed globally (for all adapters) with the parameter--overlap (or its short version -O). The option is ignored foranchored adapters since these do not allow partial matches.
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