How do I use MarkIlluminaAdapters properly in combination with its metrics for new adapters other than Illumina standards?
Hi,
We are moving our wet lab workflow (NovaSeq) from Illumina library preparation to TWIST bioscience library preparation.
In our new sample sheets for demultiplexing the samples (using bcl2fastq tool), we now needs to provide two adapters from TWIST. My question is, should I feed those adapters in our GATK pipeline under MarkIlluminaAdapters using those options?
"
--ADAPTER_TRUNCATION_LENGTH 45 --ADAPTERS null \
--FIVE_PRIME_ADAPTER "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA" \
--THREE_PRIME_ADAPTER "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT"
"
Also, would I expect a normal distribution for the read_cont from the metrics' file? To explain this point, here is what I get when using MarkIlluminaAdapters after preparing the library with illumina reagents:
## HISTOGRAM java.lang.Integer
clipped_bases read_count
4 2
9 2
10 17
11 51
12 323
13 101
14 28
15 9
16 1
36 1
But, when I trim the adapters as explained in the first point (using the options mentioned above), I get these results in the metrics file:
6 2
7 1
8 1
9 2
10 8
11 28
12 436
13 148
14 202
15 2407
16 46
17 142
18 18
19 30
20 17
21 7
22 12
23 7
24 3
26 1
27 7
28 2
29 7
30 4
31 2
32 7
33 10
34 3
35 3
37 4
38 1
39 4
40 1
41 4
42 2
43 3
44 2
45 1
46 3
48 1
49 4
50 2
51 5
52 1
53 3
54 5
55 3
56 3
57 4
58 1
59 4
60 3
61 3
62 3
63 2
64 1
66 3
67 1
68 1
69 1
70 3
71 4
72 3
74 2
76 2
77 4
79 1
80 1
86 1
87 1
88 1
92 2
99 1
101 2
105 1
110 1
Hope someone can help me understand what is right to do here.
Thanks in advance
Nawar
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Hi NawarDalila,
The GATK support team is focused on resolving questions about GATK tool-specific errors and abnormal results from the tools. For all other questions, such as this one, we are building a backlog to work through when we have the capacity.
Please continue to post your questions because we will be mining them for improvements to documentation, resources, and tools.
We cannot guarantee a reply, however, we ask other community members to help out if you know the answer.
For context, check out our support policy.
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Dear Genevieve Brandt,
Thanks a lot for your reply. My question was indeed about MarkIlluminaAdapters' abnormal results. Maybe I was not clear enough in that so here I try again:
Are those those options enough for adding new adapters to be marked by the tool? The options are:
"--ADAPTERS null \
--FIVE_PRIME_ADAPTER "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA" \
--THREE_PRIME_ADAPTER "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT"
"Otherwise, I will be waiting for the backlog :)
Best,
Nawar
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Hi NawarDalila, I will look into this request further to find out if we have any advice.
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Hi NawarDalila,
The adapters you list seem to be very short, and they seem to only have the illumina-adapter part of the oligo rather than including the entire adapter with the sample-barcode and the primer. For example, the dual indexed Illumina adapter that is included in Picard is:
AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT
and
AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
(where the N's are indicating the location and length of the sample-index.)
So I'm wondering if you have provided the complete adapter. The amount of long ends the were clipped indicates that too many bases are being clipped.
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Hi Yossi Farjoun,
Thanks a lot for your reply. If you take a look at the original website for the company (https://www.twistbioscience.com/resources/protocol/unique-dual-index-sequences-protocol-reference-document-spreadsheet-and-sample), then press "Download the Sample Sheet Templates". You can see that they specified those exact adapters in the Sample Sheet for Novaseq6000 standard (The short one that I posted). Usually we don't specify those when using the kit from Illumina (I anticipate it is automatically done as standard).
But from your answer, I know now that it will not matter because it is the same from each end. The sequence of the DNA regions will always start after the Ns in the second stage.
Thanks a lot again, your answer was very helpful and insightful.
Best,
Nawar
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